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1969

Purification and properties of [alpha]-actinin fromrabbit striated muscleRichard Morris RobsonIowa State University

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This dissertation has been microfihned exactly as received 69-15,642

ROBSON, Richard Morris, 1941-PURIFICATION AND PROPERTIES OF a -ACTININ FROM RABBIT STRIATED

MUSCLE.

Iowa State University, Ph.D., 1969 Biochemistry

University Microfilms, Inc., Ann Arbor, Michigan

PURIFICATION AND PROPERTIES OF a-ACTININ

FROM RABBIT STRIATED MUSCLE

by

Richard Morris Robson

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major Subject: Biochemistry

Approved:

In Charge of Major Work

''Head of Major Department

Dean^f Graduate Collegq

Iowa State University Ames, Iowa

1969

Signature was redacted for privacy.

Signature was redacted for privacy.

Signature was redacted for privacy.

il

TABLE OF CONTENTS

Page

INTRODUCTION 1

REVIEW OF LITERATURE 4

MATERIALS AND METHODS 18

RESULTS 30

DISCUSSION 109

SUMMARY 121

CONCLUSIONS 125

LITERATURE CITED 127

ACKNOWI.EDGEMENTS 133

1

INTRODUCTION

In the past five years several'newly discovered myofibrillar proteins

have been described. Until these reports, myofibrils in mammalian striated

muscle fibers were thought to be composed almost entirely of three pro­

teins: actin, myosin, and tropomyosin. Now a-actinin, p-actinin, troponin,

and perhaps even several other as yet uncharacterized proteins introduce

new and unknown properties to the already complex nature of the contractile

apparatus.

Little is known about the protein a-actinin. Whether this protein

plays a purely structural role, or whether it somehow takes part in regula­

tion of the actin-myosin interaction, or both, is still entirely unclear.

One of the difficulties in ascertaining the role of this protein is that

presently used methods of preparation seem to result in varying degrees of

purity. Recent evidence indicates that CX-actinin may make up only five to

twenty per cent of the total protein in many so-called "Cc-actinin extracts".

Studies on the composition of the Z-line have recently suggested that

a-actinin, or a-actinin plus some other unidentified substance or sub­

stances, may be the principal components of the Z-line. Tropomyosin had

previously been thought to be the principal component of the Z-line. The

Z-line in muscle is important not only because it is the major structural

matrix holding actin filaments in proper juxtaposition, but also because

of its often abnormal appearance in several muscle diseases. Furthermore,

several recent theories of muscle contraction have proposed a much more

active role of the Z-line in the contraction process than has usually been

supposed. Thus, a-actinin and its possible physiological function in the

2

myofibril ore quite Intriguing from several points of view.

This study investigates the preparation and purification of a-actinin

and attempts to examine some properties of the protein in relation to its

interaction with other myofibrillar proteins and its possible location in

the Z-line.

Limitations of the Study

In view of the complexities of the myofibrillar protein interactions,

it was not possible to examine all possible interactions between a-actinin

and the other myofibrillar proteins. Emphasis was placed on the interac­

tion between a-actinin and actin, the influence of «-actinia on the acto-

myosin interaction as measured by the turbidity and AïPase reactions, and

to a limited extent, the possible location of a-actinin in the Z-line.

In view of the purity of a-actinin preparations made according to

previous reports, emphasis was put on the preparation of a more homogen­

eous a-actinin before further study of its properties was attempted.

Nomenclature and Abbreviations Used in the Dissertation

In this dissertation the term "actomyosin" will be used to refer to a

mixture of actin and myosin reconstituted from purified actin and purified

myosin. 'iTie words "natural actomyosin" and "myosin B" may be used synon­

ymously and will be used to refer to a mixture of actin and myosin result­

ing from a 24-hr extraction of minced muscle with a high-ionic strength

(7^/2 about 0.6) salt solution.

The following abbreviations will be used throughout this dissertation:

ATP, adenosinetriphosphate; ITP, inosinetriphosphate; EDTA, ethylene-

3

diamihetetraacetic acid; EGTA, 1,2-bi8-(2-dicarboxyinethylaminoethoxy)-

ethane; Tris, tris-(hydroxymethyl)-aminomethane; Pi, inorganic phosphate;

TV2, an ionic strength calculated on a molarity basis; DEAE, diethylamino-

ethyl; SDS, sodium dodecyl sulfate.

4

REVIEW OF LITERATURE

The contractile apparatus in sttiated muscle has been shown by

Huxley (1953, 1957, 1963) to be made up of a double array of interdigitat-

ing thick and thin filaments. The thin filaments are attached at one end

to the Z-line (Z-band) and extend between the thick filaments at the other

end. The thick filament is composed almost entirely of the protein, myosin,

whereas the thin filament is composed partly of the other major myofibril­

lar protein, actin. The interaction between actin in the thin filaments

and myosin in the thick filaments appears to be the primary event leading

to muscle contraction (Szent-Gyorgyi, 1951). Although much is known about

the myosin and actin molecules individually, the regulation of their inter­

action and the actual interaction process resulting in contraction or re­

laxation is only partly understood. There has, of course, been consider­

able effort directed toward an understanding of the actin-myosin inter­

action and even though no unifying theory is yet available, a substantial

amount of data has been accumulated on certain characteristics of this in­

teraction.

One of the characteristic features of the actin-myosin interaction is

the unique changes it causes in ATPase activity. Myosin is an ATPase

(Engelhardt and Ljubimova, 1939) which is activated by Ca^ but inhibited

by Mg^. Interaction of myosin with actin causes a striking transformation

-H- -H-of the Mg -inhibited enzyme into a Mg -activated complex (Banga and

Szent-Gyorgyi, 1943). Magnesium activation of myosin ATPase occurs only at

low (< 0.2) ionic strengths where the myosin exists as an actomyosin com­

plex in the presence of ATP. As the ionic strength is raised, the inter-

5

action between actin and myosin is weakened (Tonomura and Yoshimura, 1960;

Leadbeater and Perry, 1963) and the system again assumes the characteris­

tics of the myosin enzyme. The ATP level is also quite important with

I I higher levels of ATP favoring dissociation and Mg -inhibition. At ATP

concentrations of 3-5 mM, the KCl concentration necessary to observe

-H-appreciable Mg -activation of the ATPase activity of a purified actin-

myosin mixture lies between 20-60 mM.

A second characteristic feature accompanying the interaction of actin

with myosin is acquisition of the ability to undergo remarkable physical

changes upon the addition of ATP at low (< 0.15) ionic strength. Neither

myosin nor actin suspensions by themselves in 30-100 mM KCl exhibit any

visible change upon addition of ATP, but a suspension of actomyosin under

the same conditions undergoes a remarkable "clumping" and increase in

turbidity upon the addition of ATP. This process has been called "super-

precipitation" or "synaeresis" (Szent-Gyorgyi, 1951) since it ostensibly

involves, at least in part, a loss of water of hydration from the actin-

myosin complex. If the KCl concentration is high (80-150 mM) or ATP is

added in large amounts (3-10 mM), the actomyosin suspension will exhibit

a decrease in turbidity, called "clearing" (Spicer, 1952). Superprecipita-

tion and clearing of actomyosin suspensions resulting from ATP addition

have been regarded as an in vitro model of contraction and relaxation, re­

spectively. A simple method for monitoring these two processes was

developed in the early I960's by Ebashi (Ebashi, 1961). This test which is

widely used to study the interaction of actin and myosin, consists of fol­

lowing light absorption by an actomyosin suspension before and after ATP

addition. Depending on the ionic strength and the concentration of ATP,

6

ATP addition first causes a decrease in absorbance, this decrease usually

being interpreted as the "clearing" phase or dissociation of the actin-

myosin complex. Then after hydrolysis has lowered the ATP level to a point

where the ATP can no longer dissociate the actin-myosin complex, the system

superprecipitates, causing a rapid rise in the absorbance due presumably

to light scattering. The physical theory underlying the optical situation

in this system is not well understood, and the actual interpretation of

the results in terms of the actin-myosin interaction and the contraction

process remains a source of considerable controversy (Endo, 1964; Yasui

and Watanabe, 1965; Levy and Fleisher, 1965a,b; Tonomura et al., 1966;

Tokiwa £t al., 1967 ; Tada and Tonomura, 1967 ; Chaplain, 1967 ; Briskey

^ al., 1967a). For example, although the clearing response observed in

Ebashi's test presumably indicates a dissociation of the actin-myosin com­

plex and should therefore be accompanied by a loss of Mg'^-modified ATPase

activity, Eisenberg and Moos (1967) have recently shown that it is possible

to demonstrate an increase in the Mg^-modified ATPase activity of an

actomyosin suspension under conditions where there is no measurable increase

in turbidity. Thus, it is evidently possible for actin to modify myosin's

ATPase activity before it interacts to produce the characteristic turbidity

response. Whatever the correct interpretation, Ebashi's turbidity test is

a sensitive measure of differences in the actin-myosin interaction.

Careful study of the characteristic turbidity and Mg -modified ATPase

activities of actomyosin suspensions led directly to the discovery of a new

class of myofibrillar proteins called the regulatory proteins, because they

appear to have the ability to control or regulate the interaction of actin

and myosin in the presence of Mg and ATP. The discovery of the first of

7

these regulatory proteins was presaged by the finding that both the super-

-H-precipitation and Mg -modified ATPase activities of myosin B suspensions

were critically affected by minute amounts of Ca^ (Weber, 1959; Ebashi,

1960, 1961). Myosin B is a particular kind of actomyosin preparation made

by extraction of minced muscle with a high ionic strength salt solution for

24 hr. Since it is a direct extract of muscle, it presumably should con-

"r " " tain all the contractile proteins existing in muscle and may be contrasted

to actomyosin reconstituted from purified actin and purified myosin. By

-H-the use of EGTA, a chelating agent with a high binding constant for Ca

but a low binding constant for Mg^, Weber (1959) and Weber et al. (1964)

showed that myofibrils, which like myosin B contain all the contractile pro­

teins, required trace (10 ̂ -10 ^ M) amounts of Ca"*"*" for both maximum

-H- -H-synaeresis and a high Mg - modified ATPase activity. Removal of Ca to

10 ^ M resulted in a loss of both of these characteristic features of the

-H-actin-myosin interaction, even though Mg , KCl, and ATP concentrations were

all optimal for the interaction. This suggested that trace amounts of

Ca"^ were necessary for the actin-myosin interaction to occur in the myosin

-H-B or myofibril systems. Interest in the role of Ca in contraction was

heightened by Ebashi*s (Ebashi, 1961; Ebashi and Lipman, 1962) discovery

that the. so-called relaxing factor system in muscle was in fact nothing

more than pieces of sarcoplasmic reticulum which possessed the ability to

accumulate Ca"^ against a concentration gradient. Thus, it appeared that

-H-the release of trace amounts of Ca from the sarcoplasmic reticulum could

I "f" serve as a trigger for the contraction process, and rebinding Ca to the

sarcoplasmic reticulum membranes would effect relaxation.

Although this work in the late 1950's indicated an important role for

8

I I Ca in muscle contraction, the mechanism of calcium's action remained un­

known, It was known that actomyosin reconstituted from purified actin and

purified myosin was insensitive to the relaxing action of chelating agents

(Perry and Grey, 1956; Weber and Winicur, 1961; Maruyama and Gergely,

1962a,b; Ebashi and Endo, 1964) and thus did not require Ca"^ for synaeresis

or high Mg^-modified ATPase activity. This difference between myosin B

and myofibrils on the one hand, and reconstituted actomyosin on the other,

led to the discovery by Ebashi (1963) of a third protein component partici­

pating in the superprecipitation of actomyosin. Ebashi called this third

component, "native tropomyosin", because of the many similarities between

it and tropomyosin, a myofibrillar protein of unknown significance dis­

covered in 1946 by Bailey (1948). Both the myosin B and the myofibril sys­

tems contained "native tropomyosin", but this component was absent from

the reconstituted actomyosin system. Addition of "native tropomyosin" to

the reconstituted actomyosin system, however, made this system sensitive to

-H- -H-Ca . Thus, the Ca -sensitivity of myosin B and myofibrils was due to

the "native tropomyosin" contained in these systems.

Intensive study into the properties of "native tropomyosin" soon re­

vealed that "native tropomyosin" was composed of two different proteins

(Ebashi and Kodama, 1965). One of these was tropomyosin itself, but the

other was a hitherto undiscovered component of the myofibril. Ebashi

called this new protein, troponin. Troponin binds strongly to tropomyosin

and possesses a tropomyosin-aggregating effect (Ebashi and Kodama, 1965),

but it does not bind directly to myosin (Kominz and Maruyama, 1967). In

the presence of tropomyosin, troponin also exerts a pronounced effect on

the physicochemical properties of F-actin (Ebashi and Kodama, 1966), but

9

the effects of troponin alone on F-actin remain unclear. Since tropomyosin

does bind strongly to F-actin (Ebashi, 1963; Ebashi ejt al,, 1968), it now

appears that tropomyosin serves as a "cement"to hold troponin onto the F-

actin filament. This supposition is supported by Ebashi's finding that in

the myofibril troponin is considerably more labile to tryptic hydrolysis

than tropomyosin, whereas in solution, the two proteins possess similar

sensitivity to trypsin (Endo et £l., 1966; Ebashi ejt , 1968). It is

clear that troponin is the protein responsible for conferring Ca^-sensi­

tivity on the actomyosin system, and it has recently been shown that

troponin possesses both a high affinity and capacity for calcium ions

(Ebashi e^ al., 1967 ; Yasui e^ al., 1968). By using fluorescent protein

and antibody techniques, Endo a^. (1966) and Ohtsuki et al. (1967) have

shown that troponin and tropomyosin are present along the entire length of

the thin (actin) filament.

The discovery of a-actinin (Ebashi et al., 1964) followed soon after

and was closely related to the discovery of "native tropomyosin". Again,

a-actinin's discovery was partly due to differences between the properties

of myosin B and actomyosin suspensions. It had been known for some time

that under well-defined ionic conditions, addition of ATP to actomyosin

suspensions resulted in a longer clearing response than addition of ATP

under the same conditions to a myosin B or myofibril suspension (Ebashi

et al., 1964). Analytical ultracentrifugal examination of the same "crude

extract" of muscle residue used for preparation of "native tropomyosin" re­

vealed the presence of a second protein fraction (Ebashi e^ a^., 1964).

This second protein fraction, when added to a reconstituted actomyosin sus­

pension, greatly accelerated the onset of superprecipitation. Thus, the

10

second protein appeared to possess the ability to enlarge the range of

ionic strength and ATP concentration in which actomyosin will superprecipi-

tate in response to ATP. Since living muscle operates in an ionic strength

(0.15-0.18) and ATP concentration (5 inM) that are much higher than those

ordinarily used for in vitro experiments, Ebashi e;t al^., (1964) suggested

that this second protein may play a fundamental role in muscle contraction

by enabling the actin-myosin interaction to occur under conditions where

this interaction would not occur in its absence. Ebashi and Ebashi (1965)

named this second protein "a-actinin" both because of the similarity of its

amino acid content to that of actin, and because of its activating effect

on the actin-myosin interaction.

The preparation and many of the properties of the a-actinin fraction

were described in two early papers (Ebashi and Ebashi, 1965; Maruyama and

Ebashi, 1965). Briefly, a-actinin was prepared from a myosin-extracted

residue which had been rinsed four times in low ionic strength solutions at

2°C and subsequently extracted with water at room temperature for several

hours. The watery extract was collected by filtration. When subjected to

ammonium sulfate fractionation, the à-actinin fraction was precipitated at

approximately 20 per cent saturation whereas "native tropomyosin" precipi­

tated between approximately 40-70 per cent saturation. Purification of the

a-actinin extracts (when attempted) was done primarily by repeated ammonium

sulfate fractionation. Besides the apparent influence of a-actinin on the

superprecipitation of actomyosin, a-actinin was shown to interact with F-

actin (polymerized or fibrous) and not with G-actin (depolymerized or

globular) or with myosin. However, the G-to-F actin polymerization was

considerably accelerated by the presence of a-actinin. The addition of

11

Ot-actinin to F-actin caused n gelation and, at high a-actinin to F-actin

ratios, a precipitation of actin. Maruyama and Ebashi (1965) indicated

that their results suggested that a-actinin caused a cross-linking of F-

actin strands, but flow birefringence measurements failed to demonstrate

any marked increase in average filament length upon addition of a-actinin

to F-actin, and indeed even suggested a slight decrease in average molecular

length at high a-actinin to F-actin ratios. ' I" [ •

The effect of a-actinin on the Mg -modified ATPase activity of acto­

myosin was first studied by Maruyama (1966a). When he used a-actinin pre­

pared by Ebashi's procedure, Maruyama found that a-actinin did not lead to

an increase in Mg -modified ATPase•activity of actomyosin suspensions un­

less large amounts of a-actinin were used. At a-actinin to F-actin ratios

of 10:1, the KCl concentration where clearing occurred was shifted to higher

KCl concentrations. Also, p-actinin, a third regulatory protein, first

isolated and characterized by Maruyama and co-workers (Maruyama, 1965;

Maruyama, 1966a,b; Maruyama and Kawamura, 1968) had no effect on the Mg -

modified ATPase activity of actomyosin suspensions at any of the P-actinin

to F-actin ratios studied. p-Actinin apparently functions to regulate or

control the length of F-actin filaments at approximately 1 p although this

role is not entirely clear at present (Maruyama and Kawamura, 1968).

In their first description of the discovery of a-actinin, Ebashi

et al. did not clearly describe the physical properties of a-actinin,

(Ebashi ejt £l., 1964) except to mention that in the absence of salt its

S-rt was about 10. In 0.1 M KCl, this value increased to more than 40. 20,w

Ebashi interpreted this as a remarkable salt-induced aggregation, but since

the intrinsic viscosity of the solutions remained low even in the presence

12

of salt, the aggregation was apparently a random process. Subsequently,

Ebaahi and Ebashi (1965) lowered their estimate of the sedimentation co­

efficient of a-actinin to 6S and also published the first amino acid analy­

sis of a-actinin showing its remarkable similarity to the amino acid com­

position of actin. Since the possibility clearly existed that a-actinin

was simply a denatured form of G-actin, Ebashi and Ebashi (1965) denatured

G-actin in several ways, but they could find no contraction enhancement

properties in any of the denatured G-actin preparations.

Soon after Ebashi's initial report, the preparation and properties of

a-actinin were reinvestigated by Briskey and associates (Briskey et al.,

1967a,b; Seraydarian et , 1967, 1968) in an extensive study using many

of the same procedures described in Ebashi's original reports. For the

most part; Briskey's findings closely agreed with those of Ebashi. Briskey

and associates placed considerable emphasis on the point that a-actinin

should not be identified as a required factor in the contraction process

but that myosin and F-actin purified free of a-actinin would still give a

turbidity response and a high Mg -modified ATPase activity, both indica­

tive of contractile activity. In fact, Ebashi and Ebashi (1965) had made

this same point in their earlier work when they indicated that it was pos­

sible to find conditions where actomyosin would give a contractile response

in the absence of a-actinin and had simply suggested that a-actinin en­

hanced contraction, particularly at ionic strengths closer to the physio­

logical range. Briskey and associates demonstrated that although a-actinin

considerably enhanced the turbidity response of actomyosin at low ionic

strength, this enhancement disappeared at physiological ionic strength.

Briskey et £l. (1967b) also reaffirmed Maruyama and Ebashi's (1965) earlier

13

finding that a-actinin caused "gelation" or "crosslinking" and reported

that the binding ratio of a-actinin to F-actin was 0,9 to 1.0, This ratio

was later shown by work in the same laboratory to be incorrect (Goll et al^, ,

1969). However, Briskey et (1967b) did differ from Ebashi's earlier

work in finding an interaction between G-actin and a-actinin. The pro­

nounced ability of a-actinin to crosslink F-actin strands led Briskey and

associates to propose that a-actinin represents Z-line material; however,

no evidence was presented in support of this hypothesis.

The purity of the a-actinin preparations used by Briskey and asso­

ciates is open to considerable question. The preparations were presumably

very similar to those of Ebashi and Ebashi (1965) since almost identical

methods were used in the two studies. Briskey and associates (Seraydarian

et al., 1967) showed that their a-actinin migrated primarily as a single

peak in a low ionic strength solution in the analytical centrifuge, but

later (Briskey e^ £l•, 1967b) mentioned that, in fact, indications of more

rapidly sedimenting material were present. Furthermore, the analytical

ultracentrifugal patterns shown were of a preparation containing only

2.2 mg protein/ml, a concentration so low that it is very difficult to

detect inhomogeneity in the analytical centrifuge.

Soon after Briskey's reports, Nonomura (1967), working in Ebashi's

laboratory, did demonstrate the presence of considerable inhomogeneity in

ft-actinin made by Ebashi's (or Briskey's) methods. By simple differential

centrifugation with a preparative ultracentrifuge, Nonomura succeeded in

separating Ebashi's a-actinin extracts into three components, although

careful examination of Nonomura's evidence suggests that even these com­

ponents were not homogeneous. The s of the three components were

14

6.2, 10.0 and 25.6. Both the separation and the sedimentation coefficients

were again determined in very low ionic strength solution. All three com­

ponents appeared to have similar accelerating effects on the superprecipita-

tion of actomyosin. However, this observation has since been refuted, and

it now appears that only the 6S component possesses O^actinin activity

(Drabikowski £t al_. , 1968; Coll £l., 1969).

Drabikowski and co-workers (Drabikowski ct. fi- » 1968; Drabikowski and

Nowak, 1968) have recently reported that tropomyosin appears to alter the

interaction between actin and a-actinin. Their evidence indicates that

tropomyosin abolishes the gelation and precipitation of F-actin caused by

high levels of CC-actinin. Drabikowski again emphasized that the purity of

Ebashi's (or Briskey's) a-actinin preparations, even after Nonomura's differ­

ential sedimentation purification, was open to considerable question. The

specific activity of the Q!-actinin extracts in Drabikowski's study appeared

to vary substantially as measured by the relative gelation effect of a

given amount of o^actinin on F-actin. Because of this, it. was impossible

to determine the stoichiometry of the interaction between o;-actinin and

actin.

In 1967, Goll et (1967) presented the first evidence in confirma­

tion of Briskey's suggestion that CC-actinin may be a constituent of the

Z-band of the myofibril. Goll found that limited trypsin digestion causes

a very rapid liberation of a protein possessing CC-actinin activity from

myofibrils. The liberation of CC-actinin activity coincides with structural

deletion of the Z-band. Later that same year, Masaki £t ^al. (1967) inde­

pendently confirmed Coil's finding when they found that antibody prepared

against a-actinin will bind only the Z-band.

15

Before these reports, it was generally felt that the Z-band consisted

primarily of tropomyosin. This feeling was based on three general lines of

evidence: 1) the fact that selective extraction of actin and tropomyosin

resulted in disappearance of the Z-line from myofibrils (Corsi and Perry,

1958; Corsi £t al., 1967), 2) the finding that fluorescent antibodies pre­

pared against tropomyosin were bound to the Z-line as well as the thin fila­

ment (Endo e^ £l., 1966); the evidence on this point, however, was not very

clear, and 3) Huxley's (1963) report that the ultrastructure of tropomyosin

crystals- closely resembles the ultrastructure of cross-sections through the

Z-line.

A major advance in the study of Z-line composition was made by Stromer

and co-workers (Stromer et , 1967, 1969) who discovered a method for

first extracting the Z-line from glycerinated rabbit psoas muscle, and then

by incubation with fractions of the extracted protein under proper ionic

conditions, to reconstitute this structure in the extracted myofibrils.

Z-line extraction was accomplished by teasing the psoas muscle bundles into

very thin fiber bundles and then incubating the teased fiber bundles with a

low ionic strength solution for several days. A 40 per cent ammonium sul­

fate fraction of the extract obtained from the teased myofibrils was found

to reconstitute the Z-line structure in the extracted myofibrils. In addi­

tion to the material bound to the Z-line region, incubation with this Pq

fraction caused the formation of small cross-bridges between adjacent thin

filaments as well as the appearance of tufts of material resembling the re­

stored Z-line material in the lateral thirds of the I band. This 40 per

cent fraction was shown to contain no detectable tropomyosin. Likewise,

fractions containing tropomyosin did not restore Z-lines. a-Actinin

16

preparations made according to Ebashi's method resulted in strong cross-

bridge formation, but either no or only a slight binding reaction in the Z-

line. No evidence was presented concerning the a-actinin activity of the

a-actinin preparations used. Stromer e^ £l. (1969) also showed that, al­

though ultrastructure of the Z-line and of tropomyosin crystals were very

similar, there were distinct structural differences between the two. This

1 has recently been confirmed by Caspar £t by using x-ray diffraction

techniques.

Thus, it presently appears that tropomyosin is not a primary consti­

tuent of the Z-line, but since Stromer e_t £l. could not obtain Z-line re-

constitution with Ebashi's a-actinin extracts, the exact role of a-actinin

in Z-line structure also remains unclear. Since Nonomura (1967) had shown

that Ebashi's a-actinin is inhomogeneous, it is possible that the a-actinin

preparations examined by Stromer e_t al. contained too little a-actinin to

be effective in Z-line reconstitution. This possibility is made more proba­

ble by Coil's (Coll £t al^. , 1969) recent suggestion that as much as 80-95

per cent of Ebashi's (or Briskey's) a-actinin preparations was inactive

protein. This suggestion was based on the finding that when "partially

purified a-actinin" extracts (Seraydarian et al., 1967) were subjected to

extensive tryptic digestion almost all of this original a-actinin activity

could be recovered in a small fraction making up only 6 per cent of the

original protein. Furthermore, the sedimentation coefficient of the protein

in this small fraction was about 6.3S which is very close to that of

^Caspar, B, L. D., C. Cohen and W. Longley, Children's Cancer Research Foundation, Boston, Massachusetts. X-ray diffraction of tropomyosin crystals and the Z-line. Private communication. 1969.

17

the major protein in the orlginnl extract. The sedimentation di,'igr;mi and

o:-actinln activity of this protein (called A-protein l)ecause it came from

a-actinin) closely resembled that of the protein isolated after brief

tryptic digestion of myofibrils. The latter protein was called Z-protein,

because it presumably came from the Z-line. The amino acid composition of

the Z-protein was clearly different from that of CC-actinin as reported by

Ebashi and Ebashi (1965) and therefore also different from that of actin.

On the basis of this evidence, Goll et al. suggested that Ebashi's a-actinin

extract may contain between 80-95 per cent denatured G-actin. This would

account for both the KCl-induced aggregation of Ebashi*s a-actinin extracts

(Katz, 1963) and the close resemblance in amino acid composition between

Ebashi's a-actinin and actin.

Clearly, further elucidation of the properties and role of cx-actlnln

in muscle will depend on the successful preparation of a more homogeneous

a-actinin, and this was one of the primary aims of this study.

18

MATERIALS AND METHODS

Except where noted, preparation of all samples was performed at 0-3°C,

All solutions in this study were prepared with double-deionized, distilled

water that had been redistilled in glass and stored in polyethylene con­

tainers. All reagents were of the highest grade obtainable.

Rabbits used for muscle samples were given sodium pentobarbital (90

mg) and d-tubocurarine chloride (1.5 mg) prior to exsanguination. Muscles

were excised immediately, immersed in ice, and then trimmed free of fat and

connective tissue, ground in a pre-cooled meat grinder, and used directly

for various protein preparations.

Myosin was prepared according to the method of Seraydarian e^ al.

(1967) .

a-Actinin-free actin was prepared according to "Scheme II" of

Seraydarian e^ (1967) .

Actomyosin was made by mixing one part actin to two parts myosin by

weight in 400 mM KCl. The mixture was stirred, diluted to 100 mM KCl, and

centrifuged at 1,000 x g for ten min. The precipitate was adjusted to

400 mM KCl and dissolved. The precipitation and dissolution steps were re­

peated once. Actomyosin was always made as soon as possible, and usually

within one hour, after fresh myosin and actin became available.

"Partially purified a-actlnin" was prepared according to "Scheme III"

of Seraydarian eit ^l, (1967). This Is essentially the procedure of Ebashi

and Ebashi (1965). It was observed that more «-actinin activity was ob­

tained if 11.4 gm instead of 10 gm ammonium sulfate/100 ml were used in

"step III" of "Scheme III". Thus, if 10 gm ammonium sulfate/100 ml were

19

used, it will henceforth be termed "partially purified a-actinin" (10 gm)

and if 11.4 gm ammonium sulfate/100 ml were used, it will be referred to as

"partially purified a-actinin" (11,4 gm). The supernatant resulting from

this step was occasionally saved and 11.1 gm ammonium sulfate/100 ml ex­

tract added. The precipitate was collected by centrifugation for 10 min at

10,000 X g, subsequently dissolved, and dialyzed against 1 mM KHCO^. The

resulting fraction was termed ^23-40'

One of the intents of this investigation was the procurement of a more

homogeneous preparation of CC-actinin for subsequent investigations. As

will be discussed later, the "partially purified CC-actinin" extracts

appeared undesirable for this purpose. Thus, a totally new procedure for

the preparation of CC-actinin was developed. Figures 1 and 2 describe the

steps used in this new procedure. Further discussion of this procedure

will be presented in the "Results" section.

Molecular exclusion column chromatography of a-actinin extracts was

done with Sephadex G-200 (Pharmacia Fine Cehmicals, Inc., Piscataway,

New Jersey), Sagavac 8F (8 per cent agarose) (Seravac Laboratories,

Maidenhead, England), and Sepharose 4R (4 per cent agarose) (Pharmacia Fine

Chemicals, Inc., Piscataway, New Jersey). Preparation and pouring of,

columns was done according to the individual company's directions. Both

ascending and descending column chromatography were used. Flow rates,

amount of sample applied, and column size will be mentioned where appro­

priate in the "Results". Sephadex columns (2.5 by 100 cm or 2.5 by 200 cm)

and accessaries were used. For both ascending and descending chromato­

graphy, Sephadex flow adapters were used for automatic sample application.

Flow rate was evenly controlled by an LKB Re Cy Chrom Peristaltic Pump

Figure 1. Flow sheet showing preparation of "swollen" myofibrils and crude 2°C-o;-actinin extracts

Muscle tissue was obtained from rabbit back and leg muscles. All extractant volumes through Step VII are based on the weight of ground tissue used in Step I.

21

Supernatant (discard)

Muscle Tissue (a) Co/ursely grind tissue (200 gm). (b) Suspend in 10 vol. (v/w) 0.25 M

sucrose, 1 mM EDTA, 0.05 M Tris-HCl, pH 7.6 by use of Lourdes homogenizer for 45 sec.

(c) Centrifuge at 1,000 x ̂ for 10 min.

II Sediment (a) Resuspend myofibrils in 5 vol.

original extraction media. (b) Centrifuge at 1,000 x ̂ for 10 min.

Supernatant (discard)

Supernatant (discard)

Supernatant (discard)

Supernatant (discard)

Supernatant (discard)

III Sediment (a) Resuspend in 5 vol. 0.05 M Tcis-

HCl, 1 mM EDTA pH 7.6. (b) Pass through a strainer to remove

connective tissue. (c) Centrifuge at 1,000 x for 10 min.

IV Sediment (a) Resuspend in 5 vol. 0.15 M KCl. (b) Centrifuge at 1,000 x for 10 min.

V Sediment (a) Resuspend in 5 vol. 1 mM EDTA. (b) Centrifuge at 1,000 x for 10 min.

VI Sediment (a) Resuspend in 5 vol. HLO. (b) Centrifuge at 2,000 x ̂ for 15 min.

VII Sediment (a) Resuspend in 5 vol. H„0. (b) Centrifuge c^t 2,000 x £ for 30 min.

22

Supernatant (discard)

îilii Supernatant (discard)

Supernatant (discard)

Supernatant (Extract A)

Supernatant (Extract B)

VIII Sediment (a) Resuspend in H„0 to a total vol.

of 2,400 ml. (b) Centrifuge at 10,000 x £ for 20 min.

IX Sediment (a) Resuspend in H„0 to a total vol.

of 2,400 ml. (b) Centrifuge at 14,000 x for 30 min.

X Sediment (a) Adjust pH of "swollen" myofibrils

to pH 8.5 with solid Tris. (b) Add 2-mercapto-ethanol to 7.13 mM

final concentration. (c) Store at 2% for 64-72 hr. (d) Centrifuge at 53,700 x ^ for 1 hr.

XI Sediment. (a) Resuspend in same vol. of 1 mM

Tris-HCl, pH 8.5, as obtained in extract A.

(b) Centrifuge at 53,700 x ̂ for 1 hr.

Sediment (discard)

Figure 1 (Continued)

Figure 2. Flowsheet showing ammonium sulfate fractionation of "Extracts A and B" from Figure 1

24

I. Combined A + B (a) Add 8.0 gm (NH^)2SO^/100 ml extract.

Adjust pH to 7.0 with NH^OH.

(b) Let stand at 0°C for 15 min. (c) Centrifuge at 10,000 x ̂ for 10 min.

Sediment (a) Dissolve in 0.5 mM

KHCO , 7.13 mM 2-merc ap to-e thano1.

(b) Dialyze against 0.5 mM KHCO for 24-48 hr.

(c) Centrifuge at 40,000 X for 30 min.

I Sediment (discard)

Supernatant

(^0-15)

Supernatant (a) Add 6.4 gm (NH,>280^/100 ml

extract. Adjust pH to 7.0 with NH.OH.

(b) Let stand at 0°C for 15 min. (c) Centrifuge at 10,000 x for

10 min.

Sediment (a) Dissolve in 0.5 mM KHCO

(b)

(c)

3'. 7.13 mM 2-mercapto-ethanol. Dialyze against 0.5 mM KHCO. for 24-48 hr. Centrifuge at 40,000 x g for 30 min.

Supernatant (a) Add 9.9 gm (NH^)^SO^/100

ml extract.

I Sediment (discard)

1 Supernatant

(^15-25)

Adjust pH to 7.0 with NH.OH. (b) Let stand at 0°C for 15 min. (c) Centrifuge at 10,000 x g

for 10 rain.

Sediment (a) Dissolve in 0.5 mM

KHCOj, 7.13 mM 2-mercapto-ethano1.

(b) Dialyze against 0.5 mM KHCO for 24-48 hr.

(c) Centrifuge at 40,000 X g for 30 min.

Supernatant (Contains tropomyosin-troponin) .

Sediment (discard)

Supernatant

(*25-40^

25

(LKB Instruments, Inc., Rockville, Maryland), Fractions were collected in

LKB fraction collectors and results monitored by measuring absorbance with

a Zeiss PMQ II Spectrophotometer at 280 nm.

DEAE-cellulose chromatography was performed with "Cellex D", DEAE-

cellulose, exchange capacity of 0.9 meq/gm, obtained from Bio-Rad Labora­

tories, Richmond, California. The cellulose was suspended in water, and

after removal of the fine particles by décantation ten times, the material

was washed first with 0.5 N NaOH and then with 0.5 N HCl. Excess base or

acid was removed with distilled water after each of the washings. The

cellulose was equilibrated with five changes of 0.02 M Tris-acetate, pH 7.5,

allowed to stand at 2°C, and placed in a column under gravity until the

cellulose had settled; the column was then packed under one pound of nitro­

gen pressure. Column dimensions will be given with results. A Sephadex

sample applicator was placed on top of the DEAE-cellulose. The sample,

dissolved in the same buffer (0.02 M Tris-acetate, pH 7.5), was placed on

the column. After the protein solution had been absorbed on the column,

several ml of starting buffer were carefully applied to the top of the

column. The column was connected to the LKB pump and a 0-500 mM KCl

gradient was started. The gradient was made by using two beakers of

approximately equal diameters, one containing 20 mM Tris-acetate, pH 7.5,

and the other containing an equal volume of 500mM KCl, 20 mM Tris-acetate, pH

7.5. The two beakers were connected by a small siphon made of glass tubing.

The contents of the beaker initially containing 20 mM Tris-acetate, pH 7.5,

was stirred continuously by a magnetic stirrer. Some material was normally

retained on the column after the KCl gradient, and this material was eluted

with 0.5 N NaOH. Fractions were collected by an LKB fraction collector and

results were measured by monitoring absorbance at 280 nm. Potassium

26

chloride concentration in the fractions was determined by. titrating the

chloride with 0.10 N AgNO^, using potassium chromate as an internal indi­

cator.

Desired fractions from chromatographic work were pooled, and concen­

trated by either salting-out with ammonium sulfate or concentration with a

Diaflo Model 401 or Model 50 Ultrafiltration Cell (Amicon Corporation,

Lexington, Massachusetts) and a UM-10 Ultrafilter membrane. After concen­

tration, extracts were normally dialyzed against 1 mM KHCO^ for 24-48 hr

and clarified at 183, 379 x £ for 30 min. Protein concentrations were

measured by the biuret method (Gomall e^ , 1949). Where necessary,

the method of Robson e^ al, (1968) was used when protein samples con­

tained Tris. Protein concentration of dilute protein solutions was deter­

mined with the Folin-Lowry method (Lowry et , 1951) modified by Coll

(Goll et al., 1964).

Analytical ultracentrifugal studies were conducted on a Spinco Model

E analytical ultracentrifuge equipped with a RTIC unit. All runs were made

at 20.0°C using Kel-F center pieces. Plates were measured using a Nikon

profile projector.

ATPase measurements were done according to the procedure of Goll and

Robson (1966). During the course of this study, it was found that in order

-H-to obtain a consistent increase in Mg -modified ATPase activity by OL-

actinin, it was necessary to premix a-actinin and actomyosin in the desired

ratio before addition of Mg^ or diluted with water to the final protein

concentrations. This procedure also resulted in increased precision be­

tween duplicate analyses. This may be due to the fact that if added

separately, the proteins are not well mixed. All assays were conducted at

27

25.0°C and the inorganic phosphate released was measured by the method of

Taussky and Shorr (1953). The electrolyte medium will be specified in the

individual experiments.

Turbidity measurements were made primarily according to Ebashi (1961).

This was done by measuring absorbance in 1 cm glass cells with a Zeiss

Spectrophotometer PM Q II at 660 nm at 26°C. The reaction was started by

the addition of ATP. Again, it was important for consistent and reproduci­

ble results to mix the actomyosin and n:-actinin before adding the other

substances. The electrolyte medium will be specified in the description of

individual experiments.

Amino acid composition was determined in the following manner. Two mg

of protein were dissolved in 6N HCl to a total volume of 2.0 ml. The tube

containing the sample was sealed under vacuum and placed in a mineral oil

bath at 110°C,for 24 hr. The protein hydrolysate was cooled, evaporated to

dryness on a Buchler flash evaporator (Buchler Instruments, Fort Lee,

New Jersey), redissolved in 2.0 ml water and evaporated to dryness two addi­

tional times. The dried samples were sent to the Wisconsin Alumni Research

Foundation, Madison, Wisconsin, where analyses were conducted with a

Phoenix automatic amino acid analyzer.

The electron microscopy in the Z-line reconstitution experiments was

kindly performed by Dr. Marvin H. Stromer (Iowa State University, Ames,

Iowa) using the techniques he has previously described (Stromer £t ,

1967, 1969). After sectioning and staining, samples were examined in an

RCA EMU-4 electron microscope. More detailed information will be given

with the individual experiment.

The crude Z-line extracts used in this study were obtained by methods

28

similar to those of Stromer et al^. (1967, 1969). Rabbit psoas muscle which

had been glycerinated for at least 30 days in 50 per cent glycerol was

teased into thin bundles of myofibrils in 2 mM Tris, pH 7.6, 6 mM 2-

mercapto-ethanol at 2°C. After one hr the myofibrils were transferred to

fresh Tris-2-mercapto-ethanol solution and stored for 10-12 days. The

supernatant solution was removed, centrifuged at 105, 651 x ̂ for 1 hr and

the solution (crude Z-line extract) was then concentrated with a Diaflo

Model 401 Ultrafiltration Cell using a UM-10 ultrafilter. A portion of the

resulting concentrated solution was fractionated with ammonium sulfate into

two fractions, Pq (11.4 gm ammonium sulfate/100 ml solution) and

(an additional 12,9 gm ammonium sulfate/100 ml to the supernatant from

Pg 20^' precipitates were collected by centrifugation at 10,000 x £

for 10 min, dissolved in 0.5 mM KHCO^, 7.13 mM 2-mercapto-ethanol, and

dialyzed against 1 mM KHCO^ for 24 hr. The protein solutions were then

clarified by centrifugation at 40,000 x ̂ for 30 min.

The methodology used to determine the stoichiometry of F-actin and

a-actinin interaction was as follows: to a fixed amount of F-actin

(usually 20 mg), contained in a series of centrifuge tubes, purified (DEAE)

a-actinin, P^^ (DEAE), was added to each tube (Set a) in increasing

quantities. The medium contained 100 mM KCl, 20 mM Tris-acetate, pH 7.5.

The tubes were each well mixed, allowed to stand at 0°C for 30 min, and

then centrifuged at 183,379 x £ for 1 hr. ITiis effected complete sedimenta­

tion of the F-actin or the a-actinin-F-actin complex. The supernatant was

collected quantitatively from each tube, dialyzed against 1 mM KHCO^ for

18 hr and the protein concentration in each supernatant determined by the

Folin-Lowry method (Lowry et al., 1951). In a parallel set of tubes (Set b)

29

the same amounts of M-actinin were centrifuged in the absence ofF-actin

because a part of the a-actinin would sediment by itself. Thus, Set ̂

gives, for each quantity of a-actinin, the amount that would remain in the

supernatant solution with no F-actin present. The corresponding value in

Set a is subtracted from this, and yields the amount of CC-actinin bound co

actin. It is not excluded that the amount of a-actinin that would sediment

by itself (about 12-13 per cent in these experiments) may also combine with

actin if it was present, but no explicit information is obtainable on this

point. The supernatant proteins were all assayed for a-actinin activity by

using the ATPase and turbidity tests.

30

RESULTS

"Partially purified a-actinln" (10 gm) as made by Seraydarlan £t al.

(1967) was found to cause very low and variable activation of the ATPase

activity and superprecipitation of actomyosln suspensions. Furthermore,

marked heterogeneity was evident in the sedimentation diagram of this pro­

tein. Fractionation at slightly higher per cent ammonium sulfate satura­

tion "partially purified a-actinin" (11.4 gm) resulted in a slight in­

crease in yield of protein as well as slightly increased ability to acti­

vate the ATPase activity and turbidity response of actomyosln. The sedi­

mentation profile of this latter preparation in 1 mM KHCO^ Is shown in

Figure 16a. A very broad leading edge indicative of a substantial amount

of aggregates is evident, although much of the protein sedimented as two

peaks. In 100 mM KCl (Figure 16b), the preparation showed even greater

aggregation and exhibited a 65 peak (trailing peak) that was very small

relative to the total protein concentration of 6.0 mg/ml. Potassium

chloride obviously promotes aggregation of the faster sedlmenting species.

These results are in excellent agreement with those of Coll £t al. (1969)

who reported sedimentation properties Identical to these for "partially

purified a-actinln" (10 gm). Furthermore, Goll et (1969) found that

although repeated salting out with either ammonium sulfate or 3.3 M KCl

will lead to some disappearance of-the faster sedlmenting material, the

area under the schlieren-dlagram of the slowest (68) peak was even less

after this treatment than it was before. This indicates that KCl promotes

formation of a family of large aggregates in o;-actinin preparations and

suggests that attempted purification of CC-actinin extracts by repeated

31

salting-out, as suggested by Seraydarian ̂ (1967) and Ebashi and

Ebashi (1965), may not result in any substantial improvement in purity of

the preparation. Goll et ai. (1969) estimated that their "partially puri­

fied a-actinin" extracts may contain only 5-20 per cent active 6S component.

Because of these undesirable characteristics of a-actinin as prepared by

the procedures of Seraydarian et £l. (1967) and Ebashi and Ebashi (1965),

a totally new procedure was developed for the preparation of CC-actinin.

This procedure is shown in Figures 1 and 2. Essentially, the proce­

dure involves the preparation of myofibrils, washing the myofibrils in

water until they are "swollen", letting the myofibrils stand for 2-3 days

at 0-2°C and pH 8.5, and then centrifuging at a force high enough to

partially sediment them. Swelling of the myofibrils causes the extraction

of a-actinin, and the extracted a-actinin remains in the low ionic strength

supernatant obtained from the centrifugation. This supernatant can then

be fractionated with ammonium sulfate. After extensive testing, the

salting-out procedure shown in Figure 2 was adopted as giving the best

initial fractionation of the crude a-actinin extracts. The sedimentation

profiles of the Pic oo and fractions are shown in Figure 3. U-ij 13-/3 /j-4U

It was found that the fraction obtained at low ammonium sulfate levels

(Pq ^^) contained a large amount of very large aggregates and a very small

amount of 68 material. The large aggregates appeared in the schlieren

diagram before reaching speed and usually sedimented very quickly without

forming any visible boundary. This was reminiscent of "partially purified

a-actinin" preparations. Although some 63 material was usually present,

this small loss was accepted in order to increase the 6S content of the

^15 25 fraction. As shown in Figure 3a, the P^^g^ fraction contained a

Figure 3a, b. Sedimentation of

Final concentrations: a) 3.87 mg protein/ml, 1 mM KHCO ; b) 3.87 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. Temp., 20°C,

Figure 3c, d. Sedimentation of and ^25-40

Final concentrations: a) 2.80 mg PQ.^^/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.5; b) 5.00 mg ^25-40 ' mM KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

^15 —25 KHCÔ3

P|5—25 100 mM KCI

Po-15 100 mM KCI

P25-40IOO m M KCI

MINUTES AFTER

8

a)

(b)

REACHING 59,780 RPM

32 56

8 32 56

34

much higher proportion of its protein as the 6S species, and a much lower

proportion of large aggregates. Although the sedimentation profiles in

Figure 3a and b show that this fraction is obviously not homogeneous, there

is marked improvement evident when the sedimentation profiles in Figures 3a

and b are compared to those shown in Figure 16a and b for "partially puri­

fied a-actinin" (11.4 gm). In making this comparison, the relative sizes

of the 6S peaks in proportion to the protein concentrations should be

noticed. There was also a small amount of 6S species in the ^25-40

tion, but since at least two other protein species of about 8.5S and 13S

were usually present, this fraction was also discarded. It was noticed

that the S values of these two faster sedimenting species were quite similar

to those of the dimeric and tetrameric forms of phosphorylase. An enzymic

assay for phosphorylase was kindly done, by Dr. Donald Graves (Iowa State

University, Ames, Iowa), and the presence of phosphorylase enzymic activity

was confirmed in the fraction.

The reasoning which led to the development of this new procedure for

a-actinin extraction was based on two observations. First, it had been

shown by Stromer e_t ad. (1967, 1969) that Z-lines could be removed from

"teased" glycerinated rabbit psoas fibers by prolonged extraction in a low

ionic strength environment. Goll et. âl" (1967, 1969) and Masaki et al.

(1967) had presented evidence that a-actinin may be a constituent of the

Z-band, and it was therefore reasoned that this low ionic strength extrac­

tion may also solubilize a-actinin. Secondly, earlier workers (Perry and

Corsi, 1958) had shown that proteins other than myosin could be isolated

from a low ionic strength extract of myofibrils. Obviously, none of the

proteins isolaued in this early report could be identified as a-actinin

Figure 4. Effect of 25 "partially purified Qi-actinin" (11.4 gm) on activated super-

precipitation of actomyosin

I

Final concentrations: 1 mM Mg , 1 mM ATP, 100 mM KCl, 20 mM Tris-acetate, pH 7.0, ^ 0.4 mg actomyosin/ml. Actinins indicated as percent of actomyosin present. Temp., 26 C.

1.0 -

.9 -? c .8 —

O iD -7 <o -7

UJ .6 -o z < .5 -m (T o .4 —1

CO y m < .3 f

30% PARTIALLY PURIFIED

a-ACTININ 5% P,5_25

CONTROL

30% PJ5-25

14 16 18 20 22 24 26 28 30 32 34 36 TIME (MIN.)

37

Table 1. Effect of gg actomyosin ATPase^

Per cent Pi5-25 added

KCl mM 0 2 5 10 20 30

25 0.420^ 0.467 0.544 0.556 0.583 0.596

50 0.322 0.369 0.480 0.452 0.408 0.418

75 0.278 0.387 0.386 0.431 0.367 0.388

100 • 0.130 0.184 0,232 0.248 0.292 0.282

125 0.071 0.078 0.096 0.106 0.149 0.164

150 0.044 0.050 0.047 0.052 0.053 0.066

^Conditions of assay: 0.20 mg actomyosin/ral, 1 mM ATP, 1 mM MgCl„, 0.05 mM CaCl2, 20.mM Tris-acetate, pH 7.0, 25°C; Pi5_25 indicated as per cent of actomyosin present.

Figures are p moles Pi/mg actomyosin/min.

since Oi-actinin was not discovered until 1964 (Ebashi £t al., 1964). Never­

theless, a re-evaluation of this earlier work suggested that CH-actinin may

have been present in these low ionic strength extracts.

A comparison of the effects of the P^^ fraction and "partially

purified a-actinin" (11.4 gm) as made by Seraydarian, e^ al^. (1967) on the

turbidity response of actomyosin is shown in Figure 4. Five per cent of

^15 25 (calculated as per cent by weight of the actomyosin present) and

30 per cent of "partially purified a-actinin" (11.4 gm) caused an almost

identical turbidity response. Thus, the P^^ fraction appeared to be 5-6

times more effective in accelerating the turbidity response of actomyosin

suspensions than "partially purified a-actinin" (11.4 gm). When 30 per cent

38

Table 2. Effect of "partially purified a-actinin" (10 gm) on actomyosin ATPase®

Per cent "partially purified QJ-actinin" added

KCl itiM 0 2 5 10 20 30

25 0.398^ 0.411 0.438 0.412 0.436 0.459

50 0.328 0.366 0.373 0.366 0.370 0.391

75 0.285 0.300 0.311 0.335 0.354 0.312

100 0.132 0.170 0,162 0.152 0.187 0.180

125 0.072 0.076 0.076 0.078 0.100 0.103

150 0.036 0.034 0.042 0.038 0.034 0.040

^Conditions of assay: 0.20 mg actomyosin/ml, 1 mM ATP, 1 mM MgCl2, 0.05 mM CaCl2, 20 mM Tris-acetate, pH 7.0, 25°C; "partially purified a-actinin" indicated as per cent of actomyosin present.

Figures are p moles Pi/mg actomyosin/min.

^15 25 added, there was an instant turbidity response. These turbidity

responses were obtained in the presence of 100 mM KCl, a level at which

Seraydarian ejt al, (1967) found that a-actinin caused very little enhance­

ment of the turbidity response of actomyosin suspensions. It is signifi­

cant that these conditions are approaching physiological conditions existing

in muscle.

The effects of P^^ and "partially purified Œ-actinin" (10 gm) on

+4-the Mg -modified ATPase activity at various levels of KCl are shown in

++ Tables 1 and 2, Both preparations clearly cause an Increase in the Mg

modified ATPase activity of actomyosin suspensions. The difference in the

39

amount of activation between the preparations can be most readily observed

in Figure 5 which shows the per cent increase in specific activity plotted

against KCl concentration. Both actinins appear to activate the ATPase

activity at all KCl concentrations studied, with a broad peak in per cent

activation occurring about 100-125 mM. While KCl concentration has a

similar qualitative effect on ATPase activation by either of the two Cx-

actinin preparations, it is obvious that the P^^ fraction causes a

quantitatively greater effect at all KCl concentrations studied than the

"partially-purified a-actinin" fraction. When examining the specific

activities given in Tables 1 and 2, it is easy to overlook the large effect

of both actinin preparations on actomyosin ATPase activities at 100-125 mM

KCl, since larger increases in activity appear to occur at low KCl concen­

trations. However, actomyosin ATPase activity in the absence of a-actinin

is quite low at 100-125 mM KCl so an ostensibly smaller activation at these

KCl concentrations represents a larger per cent increase over the initial

activity. Thus, both actinin preparations cause the largest increase in

ATPase activity at KCl concentrations similar to those existing in the liv-

H—h ing organism. The effect of both actinins on the Mg -modified actomyosin

ATPase activity is indeed a "contraction enhancement" effect specific for

the actomyosin system since neither preparation exhibited any observable

influence on the Mg^-modified ATPase activity of myosin (Table 3). There­

fore, both actinins were affecting myosin ATPase only when it was modified

by the presence of actin. This agrees with similar observations made by

Ebashi and Ebashi (1965) and Briskey e^ (1967b).

Once a more homogeneous crude Q!-actinin preparation could be routinely

produced, attempts at further purification by using column chromatography

Figure 5. Per cent increase in actomyosin ATPase at various KCl concentrations caused by P, and "partially purified a-actinin" (10 gm)

-H-Final concentrations: 1 mM Mg , 1 mM ATP, KCl as indicated, 20 mM Tris-acetate, pH 7.0, 0.2 mg actomyosin/ml. Temp., 25°C. At each KCl concentration, varous amounts of CC-actinin were added up to 30 per cent of the actomyosin present. That level of Ct-actinin that caused the greatest increase in specific activity of Ng"*^-modified acto­myosin ATPase was used to calculate the per cent increase over the control actomyosin.

130

120

110

uiOO CO

2 90

"^80 z

uj70 CO

S60 (T

g50

^40

UJ O30

0^20

10

0 O 25 50 70 100

[kci] m m

^5-25

PARTIALLY PURIFIED a-ACTININ

i

150

42

Table 3. Effect of Q-actinin on myosin ATPase^

Per cent "partially Per cent P^^ added purified Q-actinin" (10 gm) added

0 20 0 20

50 mM KCl 0.046^ 0.045 . 0.038 0.039

125 mM KCl 0.014 0.014 0.012 0.013

^Conditions of assay: 0.20 mg myosin/ml, 1 mM ATP, 1 mM MgCl2, 0.05 mM CaCl2, 20 mM Tris-acetate, pH 7.0, 25°C; a-actinin indicated as per cent of myosin present.

^Figures are p moles Pi/mg myosin/min.

could be initiated. Such purification would have been difficult if it had

been necessary to start with the "partially purified a-actinin" preparations

containing only 5-10 per cent active material. Since a class of large

aggregates appeared to constitute the single largest contaminant, even in

the P^^ 25 fractions, molecular exclusion chromatography was the first

technique tried for further purification. Most of the protein in the

^15 25 ffBCtion was excluded from Sephadex G-200 (Figure 6), and it was

obvious that purification by Sephadex G-200 columns would be possible only

if the ratio of sample volume to column volume could be substantially re­

duced. However, the goal of this study was not only one of purity, but also

one of obtaining enough material to permit further study of its properties.

The experiments with G-200 were therefore abandoned, and attention turned

to the use of agarose gels, where more inclusion was expected. The profile

in Figure 7 shows the effluent of the P^^ fraction from a Sagavac 8F

(8 per cent agarose) column. While some separation of the large aggregates

Figure 6. The elution profile of ^ ̂ .5 by 87 cm Sephadex G-200 column

The total bed volume was 427 ml. A sample of 10.2 ml of 12.0 mg/ml was applied auto­matically. The buffer used was 20 mM KCl, 10 mM Tris-acetate, pH 7.5. The ascending flow rate was 8.4 ml per hr and 5.6 ml fractions were collected.

2.4

E 2.0

S OJ

UJ o

1.6

< m û: O CO m <

1.2

8

.4

0 50 100

=L J 150 200

m

I I >1 I I 250 300 350 400 450 EFFLUENT

Figure 7. The elution profile of 25 a 2.5 by 87 cm Sagavac 8F (8 per cent agarose) column

The total bed volume was 427 ml. A sample of 6.65 ml of 15.13 mg/ml was applied auto­matically. The buffer used was 20 mM KCl, 10 mM Tris-acetate, pH 7.0, 1 mM NaN^. The descending flow rate was 13.2 ml per hr and 4.4 ml fractions were collected.

2.2

2.0

_ 1.8

c l . 6 h

O

S ' 4

LU 1.2 o < CO oc o CO m <

1.0

.8

.6

.4

.2

50 100 150 I I I I L_

200 250 300 350 400 450 ml EFFLUENT

47

and the 6S protein was, obtained, there was still considerable overlap of

these two fractions. Sedimentation runs made on the peak included in the

gel-matrix showed some improvement in purity of the preparation since there

were fewer large aggregates visible early in the run. However, it was

found that much better separation could be obtained with Sepharose 4B

(4 per cent agarose). To improve separation even more, a very long column

(183.5 cm) was used. Figure 8 shows the profile obtained when using a long

column of Sepharose 4B to purify the fraction. Most of the large

aggregates were totally excluded and appeared at the void volume. The 6S

component was recovered from the last peak to emerge from the column. Sedi­

mentation studies of this peak after concentration showed that considerable

purification of the fraction had been achieved (Figures 9c and d).

The protein concentrations in 9c and 9d are identical thereby facilitating

direct comparison of peak size before and after chromatography. It is ob­

vious that the Sepharose-purified material contains a substantially higher

proportion of the 6S species, and that many of the large aggregates formerly

observed early in the run (0 min) have been removed. There are, however,

still a few large aggregates present in the Sepharose-purified material.

This is indicated by the gently-s loping leading edge seen early in the run.

Furthermore, if the run was conducted at high protein concentrations, a 9S

species could frequently be seen sedimenting ahead of the main 6S peak.

Effects of the Sepharose-purified protein on the turbidity and ATPase

response of actomyosin suspensions are shown in Figure 10 and Table 4, re­

spectively. Purification by Sepharose chromatography resulted in only a

slight improvement in specific activity over that possessed by the original

^15 25 fraction in either the turbidity (Figure 10) or the ATPase assay

s

Figure 8. The elution profile of on a 2.5 by 183.5 cm Sepharose 4B column

The total bed volume was 899 ml. A sangle of 6.5 ml of 15.33 mg/ml was applied auto­matically. The buffer used was 20 mM KCl, 10 mM Tris-acetate, pH 7.0, 1 mM NaN^. The ascending flow rate was 12.4 ml per hr and 6.2 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

.6 —

00 CVJ .4

UJ

0 500 700 800 100 200 300 400 600 900 ml EFFLUENT

Figure 9a, b. Sedimentation of before and after purification by DEAE cellulose

a) before, b) after. Final concentrations; 3.12 mg protein/ml, 100 mM. KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

Figure 9c, d. Sedimentation of before and after purification by Sepharose 4B

c) before. d) after. Final concentrations: 3.12 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. Temp., 20°C.

MINUTES

PURIFICATION OF P15-25

BY DEAE CELLULOSE

( a )

(b)

PURIFICATION OF P15-25

BY SEPHAROSE 4B

( c )

( d )

AFTER REACHING 59,780 RPM

Figure 10. Effect of Pi5_25 ^nd 25 (Sepharose 4B) on Mg"^-activa ted superprecipitation of actomyosin

-H- ++ Final concentrations: 1 mM Mg , 1 mM ATP, 0.05 mM Ca , 100 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.4 mg actomyosin/ml. Actinins indicated as per cent of actomyosin present. Temp., 26°C.

Figure 11. Effect of Pi5_25 and Pi5_25 (DEAE) on Mg^-activated super­precipitation of actomyosin

—h II Final concentrations; 1 mM Mg , 1 mM ATP, 0.05 raM Ca , 100 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.4 mg actomyosin/ml. Actinins indicated as per cent of actomyosin present. Temp., 26°C.

53

8

E .7

k O .6 (D (0 5 Ui

.4 o .4 z

.4 <

.3 m .3 a: .3 o

.2 CO .2 CD

.2 <

.1

0

5% P|5-25 PURIFIED BY SEPHAROSE 4Bi!

5% P|5_25

CONTROL

J L _L J L ± J L

2 4 18 20 22 24 26 28 30 32 34 36 TIME (WIN.)

.8

6 .7 c O c (P o (0

10% 1^5-25

(DEAE)

5% P|5_25 (DEAE) 15-25

(DEAE)

2% R

^CONTROL

—o

± J I L J L

12 14 16 16 20 22 24 26 28

TIME (MIN.)

54

Table 4. Effect of I'i5_25 purified by Sepharose 4B chromatography on acto-myosin ATPase^

KCl mM

P 15-25

(Sepharose 4B)

KCl mM 0 5 10 20

20 0.402^ 0,497 0.528 0,523

50 0.313 0.407 0.420 0,425

75 0.226 0.290 0.280 0.308

100 0.084 0.114 0.126 0.151

125 0.037 0.064 0.053 0.072

150 0.020 0.024 0,026 0.031

^Conditions of assay: 0.20 mg actomyosin/ml, 1 mM ATP, 1 mM MgCl2, 0,05 mM CaCl2, 20 mM Tris-acetate, pH 7.0, 25°C; Pi5_25 (Sepharose 4B) in­dicated as per cent of actomyosin present.

^Figures are p moles Pi/mg actomyosin/min,

(cf. Table 1 and Table 4), even though obvious improvement in hompgeneity

as indicated by analytical ultracentrifugation (Figure 9c,d) had been

achieved. Although this result is somewhat disappointing, it should be

pointed out that addition of CZ-actinin to an actomyosin suspension does not

always produce a response proportionate to the amount or purity of the (%-

actinin added. Because of this, it is virtually impossible to quantitate

the a-actinin assay in terms of "units" of O^actinin activity. This dis­

parity in the effects of Ol-actinin may be observed in Tables 1, 2, and 4,

where in several cases, particularly at lower KCl concentrations, addition

of a-actinin beyond a certain point produced no additional response at all.

In fact, in some experiments, addition of a-actinin past this "optimal"

55

level actually caused a decrease instead of an increase in activity. This

"optimal" level appeared to differ for different a-actinin preparations,

possibly reflecting differences in content of the 6S component. Apparently,

the only way to avoid this difficulty is to test at very low a-actinin to

actomyosin ratios where the response to Q-actinin is so small that it is

difficult to measure with any precision. This same difficulty in quantitat-

ing the CC-actinin assay was observed by both Drabikowski and Nowak (1968)

and Goll ̂ _al. (1969). Thus, one often has to describe a-actinin effects

subjectively, and in relative terms, a circumstance that makes it very dif­

ficult to compare CX-actinin content of two different heterogeneous prepara­

tions.

An additional possibility for the lack of increased specific activity

in the Sepharose-purified a-actinin is the approximately 70-hr time period

required to elute the Q!-actinin from the long Sepharose column. Since the

eluted material still had to be located in the effluent, concentrated, and

clarified, this meant that the Sepharose-purified a-actinin could not be

assayed before at least one week after initial preparation. It is a well-

known fact (Szent-Gyorgyi, 1951) that myosin and actin are highly labile

proteins that cannot be kept for longer than 7-10 days after their prepara­

tion. If the same is true of a-actinin, the long period of time required

for Sepharose purification may have resulted in some loss of specific

activity, thereby negating any increase in specific activity due to the

purification.

The amount of material which can be successfully separated by molecular

exclusion chromatography is partially dependent on the ratio of sample

volume to total column volume. Since the fraction appeared to con­

56

tain at most about 40 per cent of the 6S component, it would b(> nocessary

to apply 500 rug or more oi the 1'^^ fraction to a column if sui llcient

purified 6S protein was to be obtained to permit study of its interaction

with actin. Therefore, DEAE-cellulose columns, to which much larger sample

loads can be applied, were tested to see whether purification of a-actinin

could be achieved with ion exchange chromatography. DEAE-cellulose chroma­

tography proved to provide a very convenient and effective method for puri­

fication of the 6S component. A KCl gradient was used for elution and at a

pH of 7.5, the 6S species (peak between the vertical dotted lines in Figure

12) always was eluted between 250-300 mM KCl. The nature of the protein in

the long broad peak eluting at lower KCl concentration was not completely

elucidated, but the first part of the broad peak (100-150 ml of KCl gradi­

ent) did contain appreciable amounts of phosphorylase activity. A large

amount of the material applied to the column was very tightly bound and

could not be eluted even with 2 M KCl. This material was eluted with 0.5 N

NaOH, and examination showed that it consisted of the large aggregates

present in the fraction applied to the column. As measured by the

amount of protein eluted between the vertical dotted lines in Figure 12,

the 6S species usually made up approximately 30-35 per cent of the original

^15-25 fraction applied.

At this time, some attempts were made to purify "partially purified

a-actinin" (10 gm and 11.4 gm) by using DEAE-cellulose chromatography. The

results are shown in Figures 14 and 15, respectively. In both cases, the

peak containing the 6S species (vertical dotted lines) was eluted at exactly

the same KCl concentration (250-300 mM) as the peak containing the 6S

species from the fraction. As expected from the previous sedimenta-

Figure 12. The elution profile of 25 & 2.5 by 25 cm DEAE-cellulose column

A sample of 400 mg was applied to the column. A KCl gradient was used for initial elution and NaOH (0.5N) was used to elute tightly bound protein. The flow rate was 18 ml per hr and 6 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

E c

§ CM

UJ O z s o cn m <

0 200

I

300 400 _L i

500 600

ml EFFLUENT

05N NaOH

ADDED

OD

500

400

300 o

200i^

- 100

0 700 800 900 1000

Figure 13. Per cent increase in actomyosin ATPase caused by (DEAE) at various KCl concentra­tions

-H-Final concentration: 1 mM ATP, 1 mM Mg ,0,0 mM CaCl„, KCl as indicated, 20 mM Tris-acetate, pH 7.0, 0,2 mg actomyosin/ml, Temp,, 25°C. At each KCl concentration, various amounts of a-actinin were added up to 30 per cent of the actomyosin present. That level of a-actinin that caused the greatest increase in specific activity of the Mg'^-modified actomyosin ATPase was used to calculate the per cent increase over the control acto­myosin.

130

120-

1 1 0

w g 100 0:

90

z 80

N 70

2 60 O z 50

^ 40 w % 30 UJ Û- 20

I 0

0

P15-25 PURIFIED

BY DEAE CELLULOSE

I L 100 125

[KCD mM

Figure 14. The elution profile of "partially purified a-actinin" (10 gm) (Seraydarian et £l., 1967) on a 2.5 by 25 cm DEAE-cellulose column

A sample of 270 mg was applied to the column. A KCl gradient was used for initial elution and NaOH (0.5N) was used to elute tightly bound protein. The flow rate was 18.6 ml per hr and 6.2 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

6 —

00 CM

UJ O

CO

ifi CD <t

500

400

- 300 E 0.5 N NoOH

ADDED

y/ 900 1000 800 500 600 700

ml. EFFLUENT 400 200 300

Figure 15. The elution profile of "partially purified a-actinin" (11.4 gm) on a 2.5 by 25 cm DEAE-cellulose column

A sample of 300 mg was applied to the column. A KCl gradient was used for initial elution and NaOH (0.5N) was used to elute tightly bound protein. The flow rate was 20.4 ml per hr and 6.8 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

6 —

E 4 c O 00 CM

oW 300 400 500 600

-400

0.5N NaOH ADDED -|200.y

700 800

EFFLUENT

900 1000 lîOO

65

tion studies, these partially purified Ot-actinin extracts contained a much

lower proportion of their protein as the 6S species than did the 25

fraction, about 5-10 per cent for "partially purified a-actinin" (10 gm)

and 10-15 per cent for "partially purified a-actinin" (11.4 gm). As shown

in Figure 15, a considerable amount of protein was eluted in the first

100-150 ml after the KCl gradient was started. This material was also

shown to contain phosphorylase activity and corresponded to the 8.5S and

13S peaks normally present in "partially purified a-actinin" extracts.

Generally, 10-15-fold more phosphorylase could be obtained from the 0-40

per cent ammonium sulfate fraction of the room temperature a-actinin extract

(Seraydarian et al., 1967) than from the 0-40 per cent ammonium sulfate

fraction of the 2°C-myofibril extract used to prepare the fraction

(Figure 1). The 8.5S and 13S peaks, which have now been identified as phos­

phorylase, probably correspond to the peaks observed by Goll ejt £l. (1969)

in "partially-purified a-actinin" preparations. If the original room-

temperature extract from which partially purified a-actinin is obtained is

salted out between 23-40 per cent ammonium sulfate saturation, the protein

obtained sediments largely as 8.5 S and 13S peaks.

The DEAE-purified 6S species (concentrated from fractions between the

vertical dotted lines. Figure 12) exhibited a substantially higher specific

activity in the turbidity assay than the original P^^ ^5 fraction (Figure

11). DEAE-6S added to the extent of 2 per cent of the actomyosin present

caused a marked enhancement in the turbidity response at 100 mM KCl. Five

per cent of DEAE-6S gave a larger response than ten per cent of the original

^15 25 ff&ction. Ten per cent of the DEAE-6S species caused an almost

immediate turbidity response, and the actomyosin then very quickly underwent

66

a "clumping" process and precipitated to the bottom of the cuvette. Figure

13 shows the effect of DEAE-6S on the ATPase activity of an actomyosin sus­

pension at various KCl concentrations. The conspicuous peak in per cent

activation at 100-125 mM KCl is again evident, but the DEAE-6S fraction did

not appear to possess much more activity in the ATPase test than the orig­

inal 25 fraction.

The sedimentation profile of the P.^ fraction before and after DEAE-lo-lo

cellulose chromatography is shown in Figures 9a and 9b, respectively. The

protein concentration is identical in both cases. The increased size of

the 6S peak after DEAE purification is readily apparent. The most striking

feature of the DEAE-purification is its ability to almost completely elim­

inate the large aggregates present in the original fraction. This is indi­

cated by the absence of the broad leading edge early in the sedimentation

diagram of the DEAE-purified fraction. There is, however, an obvious though

small leading peak of 9.IS (average of 11 sedimentation runs) present in the

DEAE-6S fraction.

The S-value of this peak is higher than that of the 8.5S peak

previously identified as phosphorylase in the P^g ammonium sulfate frac­

tion. Furthermore, tests for phosphorylase activity in the 1*2.5-25 (^EAE)

fraction were negative (Dr. D. J. Graves, Iowa State University, Ames,

Iowa). The exact nature of the 9.IS species has not been determined, but

it is possible that it represents an aggregate (possibly a dimer) of the

6S-o:-actinin species, since both species came off the DEAE-cellulose column

together, and the 9.IS species was either completely absent or barely ob­

servable in the original P^^ fraction. This possibility is supported

by the observation that the 9.IS peak was not evident in low ionic strength

67

solution (Figure 21c). This latter observation also supports the contention

that the 9.IS peak is different from the 8.5 S peak of phosphorylase since

the 8.5S phosphorylase peak sediments separately from the 68 peak of

a-actinin even in 1 mM KHGO^.

Up to 500-600 mg of the 25 fraction can be applied to a 2.5 by 25

cm DEAE-cellulose column without any noticeable undesirable effects on

resolution. Purification by DEAE-cellulose was also more desirable than

agarose purification because much less time was needed to elute the 6S

species from the cellulose columns (approximately 30 hr) than from the long

column needed for agarose purification (approximately 70 hr). Thus, DEAE-

cellulose chromatography is a very effective technique for purification of

the 6S a-actinin component.

Although the amount of 6S material present in "partially-purified

a-actinin" (11.4 gm) extract was quite small (Figure 15), even this very

crude extract could be effectively purified by a single pass through a

DEAE-cellulose column. Figures 16a-d show the sedimentation profiles of

"partially-purified a-actinin" (11.4 gm) before (Figures 16a and b) and

after (Figures 16c and d) DEAE-cellulose purification. Note that 2.24

mg/ml of the DEAE-purified material produced even a larger 6S peak than

6 mg/ml of the partially purified material. The peak shown in Figure 16c

can be compared directly to the peak shown for purified a-actinin published

in Seraydarian e^ al. (1967) since identical solutions and protein concen­

trations were used. The 68 peak shown by Seraydarian e^ al. is considerably

smaller than the 68 peak shown in Figure 16c, and it is obvious that even

the "purified" a-actinin of those workers contained a considerable amount

of large aggregates. In fact, careful inspection suggests that even the

Figure 16a, b. Sedimentation of "partially purified a-actinin" (11.4 gm)

Final concentrations: a) 6.00 mg protein/ml, 1 mM KHCO^. b) 6.00 mg protein/ml, 100 KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

Figure 16c, d. Sedimentation of "partially purified a-actinin" (11.4 gm) after purification by DEAE-cellulose

Final concentrations; c) 2.24 mg protein/ml, 1 mM KHCO„. d) 2.24 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

PARTIALLY PURIFIED

«-ACTININ

PARTIALLY PURIFIED

°<-ACTININ PURIFIED

BY DEAE CELLULOSE

MINUTES AFTER REACHING 59,780 RPM

8 32 56

/()

crude 25 fraction may contain a larger proportion of the 6S species .

than the purified' fractions used by Seraydarian e^ £l. (1967). It should

be mentioned in connection with Figures 16a and c, and indeed with most of

the sedimentation diagrams shown in the literature for a-actinin, that these

runs were done in 1 mM KHCO^, a very low ionic strength solution. As

Schachman (1959) has pointed out, and as is evident from comparison of

Figures 16a and b and Figures 16c and d, sedimentation in such low ionic

strength solvents often leads to "self-sharpening" effects on the sedimenta­

tion diagram. In these studies, 100 mM KCl was routinely added to dampen

out "charge effects" and the runs shown in Figures 16a and c were done

simply to afford comparison with other reports in the literature

(Seraydarian et al,, 1967; Nonomura, 1967).

The marked improvement in the sedimentation profile of "partially puri­

fied a-actinin" (11.4 gm) caused by DEAE-cellulose chromatography was

paralleled by an improvement of the specific activity in the turbidity assay

(Figure 17). While 30 per cent of the original sample caused only a slight

acceleration of the turbidity response, 5 per cent of the DEAE-purified

fraction had a marked effect. However, 5 per cent of the (DEAE)

material appeared to be slightly more potent than the partially-purified

a-actinin (DEAE) fraction. Whether this is due to the presence of slightly

greater amounts of inactive protein in the latter fraction or to partial

denaturation of this fraction caused by its extraction at room temperature

is not known.

Since DEAE-cellulose chromatography appeared to be the most effective

purification technique for use on crude a-actinin extractions, several

studies were done using the ^5 (DEAE) fraction to determine whether

Figure 17. Effect of "partially purified «-actinin", "partially purified CC-actinin" (DEAE), and Pi5:,25 (DEAE) on Mg -activated super-precipitation of actomyosin

-H- -H-Final concentrations; 1 mM Mg , 1 mM ATP, 0.05 mM Ca , 125 mM KCl, 20 mM Tris-acetate, pH 7.0, 0,4 mg actomyosin/ml. Actinins indicated as per cent of actomyosin present. Temp., 26°C.

Figure 18. Effect of ^0-20 ^20-40 ^ Z-line extract on Mg -activated superprecipitation of actomyosin

++ -H-Final concentrations: 1 mM Mg , 1 mM ATP, 0.05 mM Ca , 100 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.4 mg actomyosin/ml. PO-20 ^20-40 fractions indicated as per cent of actomyosin present. Temp., 26°C.

72

5% P|5-25(DEAi

5% PARTIALLY PURIFIED - ACTININ (DEAE)

1-30% PARTIALLY PURIFIED «-ACTININ

CONTROL

20 22 24 26 28 30 32 34 36 38 40 TIME (mln)

30% Po-20 H - LINE EXTRACT

,30% P20.40Î-LINE EXTRACT .CONTROL

J— 20 22 24 26 28 3 0 32 34 36

TIME (mln)

73

even further purification of the 6S species could be effected by additional

chromatographic procedures. Rechromatography on a long Sepharose 4B column

produced only one, symmetrical peak in the elution profile (Figure 19).

Comparison of this elution profile to that shown in Figure 8 clearly demon­

strates the ability of a single pass through a DEAE-cellulose column to re­

move the large aggregates from crude a-actinin fractions. The peak in

Figure 19 was eluted between 600-700 ml of effluent, the same place that the

6S peak was eluted in Figure 8. Note that a 136 mg sample in Figure 19

produced a substantially larger peak than a 100 mg sample i^Figure 8. The

elution profile in Figure 19 suggests that Sepharose 4B chromatography of

the 25 (DEAE) fraction produced little or no additional purification, a

conclusion supported by examination of the sedimentation diagrams of the

P._ (DEAE) fraction before and after agarose rechromatography (cf.

Figure 9b and Figures 21a and b). There is some evidence for the presence

of a very small amount of rapidly sedimenting material in 1 mM KHCO^ (Figure

21a), but the 9.IS shoulder seen in the (DEAE) fraction appears

slightly diminished in size after rechromatography on Sepharose 4B.

Rechromatography of the (DEAE) fraction on a second similar

DEAE-cellulose-column produced a single sharp peak (Figure 20) eluting at

250-300 mM KCl, the same KCl concentration at which the 68 species is eluted

upon DEAE-chromatography of crude a-actinin fractions. The peak in Figure

20 where 72 rag of sample was applied to the column is over one-half the

size of the corresponding peak in Figure 12 where 400 mg of protein were

applied. Further elution of the column with 0.5N NaOH caused the appear­

ance of a second, small peak (Figure 20), suggesting that rechromatography

on DEAE-cellulose may eliminate a very small amount of large aggregates

Figure 19. The elution profile of 25 (DEAE) on a 2.5 by 182.0 cm Sepharose 4B column

The total bed volume was 893 ml. A sample of 11.7 ml of 11.6 mg/ml was applied auto­matically. The buffer used was 20 mM KCl, 10 mM Tris-acetate, pH 7.5. The ascending flow rate was 11.3 ml per hr and 5.65 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

2 -

0 -

8 -

6-

4-

2 -

O

8

6-

4

2

1 1 1

Ln

100 200 300 400 500 600 ml EFFLUENT

700 800 900

Figure 20. The elution profile of (DEAE) following rechromatography on a 2.5 by 25 cm DEAE-cellulose column

A sample of 71.9 mg was applied to the column. A KCl gradient was used for initial elution and NaOH (0.5N) was used to elute tightly bound protein. The flow rate was 19.8 ml per hr and 6.6 ml fractions were collected. The peak between the vertical dotted lines was used for further study.

1.2

I . I

1.0

_ 9 E «= .8 -

§7

5 .6 # 5 00 tr O 4 cn m < .3

.2

.1

A ± 0 200 300 400 500

ml

500

- 400 s

0.5N NoOH ADDED

300 1=1

200 o

100

600 700 800 900 1000 EFFLUENT

Figure 21a, b. Sedimentation of Pi5_25 after purification by DEAE-cellulose followed by chromato­graphy on Sepharose 4B

Final concentrations: a) 3.00 rag protein/ml, 1 itiM KHCO„. b) 5.00 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

Figure 21c, d. Sedimentation of (DEAE) after rechromatography purification by DEAE-cellulose

Final concentrations: c) 2.50 mg protein/ml, 1 mM KHCO^. d) 5.00 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

P|5-25 PURIFIED BY DEAE CELLULOSE

AND SEPHAROSE 4B

P|5-25 PURIFIED BY DEAE

CELLULOSE TWO TIMES

MINUTES AFTER

16

REACHING 59,780 RPM

32 72

32 72

80

still present in the (DEAE) fraction. This conclusion is supported

by the sedimentation diagrams of the DEAE-rechromatogramed material

(Figures 21c and d) . There is no evidence for the presence of any large

aggregates in either 1 mM KHCO^ or 100 mM KCl, although it was noticed that

rechromatography on DEAE appeared to cause à slight enhancement of the size

of the leading 9.IS peak.

Since rechromatography did not appear to effect any substantial im­

provement in homogeneity of the ^5 (DEAE) fraction, most subsequent ex­

periments on properties of CC-actinin were done by using this fraction. The

concentration dependence of the sedimentation coefficient of the main 6S

peak of 25 (DEAE) is shown in Figure 22, In 100 iriM KCl, 20 mM Tris-

acetate, pH 7.5, the sedimentation coefficient was only slightly concentra­

tion-dependent and extrapolated to a S^q of 6.23S. This figure is in

close agreement with the S^q of 6.28 given by Goll et ^1. (1969) for their

Z-protein, a purified form of a-actinin obtained by brief tryptic digestion

of myofibrils.

Goll £t al. (1969) found that extensive trypsin treatment of "partially

purified 0!-actinin" (10 gm) did not appreciably change the sedimentation

coefficient of the 6S species nor cause any loss in specific activity in the

ATPase and turbidity tests. A similar experiment testing the effect of

trypsin on the more purified P^^ (DEAE and Sepharose 4B) 6S species is

shown in Figures 23a and b. Forty min of trypsin treatment caused a sub­

stantial effect on the sedimentation diagram of the 6S species, with the

sedimentation coefficient of the main peak being reduced to 4.7S. In addi­

tion, a small, broad peak of slower sedimenting material (5=2,6) was evi­

dent. This slower broad peak probably represents pieces of varying sizes

Figure 22. Concentration dependence of P,_ «c (DEAE) sedimentation in 100 mM KCl, 20 mM Tris-acetate, pH 7.5. Temp., 20°C.

Graph is a least squares plot. Equation: 1/S = 0.1606 + 0.000915 Concentration.

.170

.160-5^ CO ^S=6.23

.150

CONCENTRATION (mg/ml)

Figure 23a, b. Sedimentation of (DEAE and Sepharose 4B) before and after trypsin treatment

Final concentrations; a) before trypsin treatment; 5.00 mg protein/ml, 100 mM KCl, 50 mM Tris-HCl, pH 7.6, b) after trypsin treatment; 5.00 mg protein/ml, 100 mM KCl, 50 mM Tris-HCl, pH 7.6. Temp., 20°C. Conditions of trypsin treatment: 1 part trypsin to 50 parts protein by weight, 2 parts soybean trypsin inhibitor to 1 part trypsin by weight used to stop reaction after 40 min, 100 mM KCl, 50 mM Tris-HCl, pH 7.6. Temp., 25°C.

Figure 23c, d. Sedimentation of (DEAE) in urea or SDS

Final concentrations: c) 4.00 mg protein/ml, 4M urea, pH 7.5 (incubated 30 min at 25°C. before centrifuge run). Temp., 20°C. d) 3.17 mg protein/ml, 10 mM SDS, pH 8.4 (incubated 30 min at 25°C. before centrifuge run). Temp., 20°C.

P|5-25 (DEAE AND

SEPHAROSE 4B)

TRYPSIN IZED P15-25 (DEAE

AND SEPHAROSE 48)

P|5-25 (DEAE) IN UREA

P,5_25 (DEAE) IN SDS

MINUTES AFTER REACHING 59,780 RPM

24 48 72

32 128 256

85

cleaved from the parent 6S molecule, while the 4.7S peak is probably the

core remaining after tryptic removal of these smaller pieces. It is ob­

vious that the purified 6S species has been substantially affected by tryp­

tic digestion under conditions similar to those where Goll et al (1969)

found little effect of trypsin on "partially-purified a-actinin" (10 gm).

The reasons for this discrepancy are not clear, but it is possible that the

presence of a substantial number of large aggregates in Coil's preparations

either "shielded" the 6S molecule from trypsin or provided an alternative

substrate for the trypsin. This is supported by the fact that Coil's find­

ings on trypsin sensitivity of "partially-purified a-actinin" (10 gm) have

been confirmed in this study, and furthermore, that tryptic digestion of

the crude ^5 fraction which contains a substantial amount of large

aggregates lowered the sedimentation coefficient of the main 6.2S peak in

this fraction only to 5.9S. Moreover, pH-stat studies of the tryptic diges­

tion showed that considerably more hydrolysis was occuring, particularly

during the first ten min of incubation, in the "partially purified (%-

actinin" (10 gm) or crude 25 extracts than in the (DEAE) frac­

tion, even though the 6S species underwent much less alteration in the

former preparations than in the latter.

The sedimentation profiles of P^^ (DEAE) in two denaturing solvents,

urea and SDS, are shown in Figures 23c and d, respectively. In the

presence of 4M urea, the P^^ 25 (DEAE) sedimented as two distinct boundar­

ies with sedimentation coefficients of 2.5S and 3.IS. In 10 mM SDS the

protein sedimented as one principal peak with an observed S value of 2.0.

Although shown only as a leading edge to the peak in Figure 23d, a small

leading peak with an observed S value of 2.3 could sometimes be measured in

86

the SDS solvent. The substructure of a-actinin is an almost totally unex­

plored area. Therefore, it is not clear whether this heterogeneity in de­

naturing solvents is due to the presence of two different sizes of subunits

in the a-actinin molecule or due to some heterogeneity in the original

^15 25 preparation. Nonomura (1967) reported that a-actinin in 4M

urea exhibited primarily a single 4S peak, but no schlieren diagram of the

6S species in urea was shown and the preparation Nonomura used has since

been found to contain a substantial amount of heterogeneous aggregates

(Drabikowski and Nowak, 1968).

Table 5 shows the amino acid composition of purified a-actinin, i.e.,

g _ (DEAE), or P. (DEAE and Sepharose 4B), compared to Z-protein

(Goll e_t al. , 1969), a-actinin (Ebashi and Ebashi, 1965), and actin

(Carsten, 1963) . In addition, the amino acid composition of the 1*23-40

fraction from an Ebashi and Ebashi (1965) or Seraydarian e^ £l. (1967) crude

a-actinin extract is compared to that of phosphorylase (Appleman £t ^1.,

1963).

It is somewhat difficult to compare these tabulations in detail since

the results came from so many different laboratories, but by assuming the

analyses to be technically comparable and by accepting only differences of

15 per cent or larger, purified a-actinin is higher in arg, asp, glu, ala,

and leu and lower in thr, pro, gly, ile, and tyr than Ebashi's a-actinin.

The differences for thr, glu, ile, leu and tyr are particularly marked.

Compared to actin, purified a-actinin is higher in his, arg, asp, glu, ala,

and leu and lower in thr, ser, gly, ile, and tyr, with the differences in

thr, glu, ile, leu, and tyr being particularly evident. Thus, purified

a-actinin clearly differs from Ebashi's a-actinin in 10 of the 15 amino

Table 5. Amino acid composition of purified a-actinin

Residues of amino acid per 1000 residues Amino Purified

a Z-Protein CC-Actinin Ac tin P ^

23-40 Rabbit muscle^

acid Ct-actinin P ^ 23-40

phosphorylase

Lys 53.0|l.0 26.2-0.9

(59.0-1.0)

58.4^2.2 52 5O.2J1.O 62.0 61.8 His

53.0|l.0 26.2-0.9

(59.0-1.0) 24.5-0.8 23 20.1-0.4 28.6 28.5

NH3

53.0|l.0 26.2-0.9

(59.0-1.0) (170.8) (79) (88.6-10.2) (56.6) (69.9) Arg 68.2-1.7

108.4t0.6 57.2J1.4 103.4-0.6 59.6-0.4

49 48.7-0.4 71.8 80.5 Asp

68.2-1.7 108.4t0.6

57.2J1.4 103.4-0.6 59.6-0.4

96 93.5J1.6 75.4-1.8

116.5 115.4 Thr 29.6t0.2

49.3|l.O

57.2J1.4 103.4-0.6 59.6-0.4 65

93.5J1.6 75.4-1.8 29.3 40.3

Ser 29.6t0.2 49.3|l.O 55.6-1.6

139.3:4.4 52 64.4^3,0

109.1J2 . 6 47.4 30.0

Glu 164.2Î2.3 55.6-1.6 139.3:4.4 122

64.4^3,0 109.1J2 . 6 112.3 113.8

Pro 46.9:0.8 52.5:1.0 69.8^0.7

59 50.2|2.0 74.6|3.0 80.8^0.6

50.8 42.7 Gly 62.5^0.7

96.7J1.8 44.7J0 .6 47.7J1.O

52.5:1.0 69.8^0.7 77

50.2|2.0 74.6|3.0 80.8^0.6

68.5 56.9 Ala

62.5^0.7 96.7J1.8 44.7J0 .6 47.7J1.O

87.8-1.8 80

50.2|2.0 74.6|3.0 80.8^0.6 83.6 75.1

Val

62.5^0.7 96.7J1.8 44.7J0 .6 47.7J1.O

60.0 48 49.7 Jl.2 73.2:3.2

63.8 73.1 Ile

62.5^0.7 96.7J1.8 44.7J0 .6 47.7J1.O 64.9 70

49.7 Jl.2 73.2:3.2 47.6 58.8

Leu 107.7-2.3 95.8 79 68.8^1.4 42.9|0.4

99.8 96.2 Tyr 26.2^0.3 33.5-0.9

36.5-0.7 42

68.8^1.4 42.9|0.4 39.8 43.1

Phe 35.4-0.6 33.5-0.9 36.5-0.7 35 31.3-0.6 46.2 47.1

^Purified Q!-actinin is the P]^5_25 fraction purified by DEAE-cellulose or by DEAE-cellulose fol­lowed by Sepharose 4B. Mean plus or minus standard error of analyses on four different preparations.

^From Goll et al^. (1969) .

*'From Ebashi and Ebashi (1965) .

^From Carsten (1963), Figures are means plus or minus average of the deviations from the mean.

^Mean of two analyses.

^From Appleman et (1963). Calculated after subtracting out tryptophan residues.

88

acids listed and from actin in 11 of the 15 amino amino acids listed. Goll

et al. (1969) suggested that Ebashi's cc-actinin contained a considerable

proportion of denatured G-actin and that this accounted for the remarkable

resemblance in amino acid composition between Ebashi's a-actinin and actin.

Goll et. âÀ' (1969) prepared a protein, which they called Z-protein, that

appeared to be a purer form of a-actinin than Ebashi's preparation, and

whose amino acid composition differed somewhat from either Ebashi's (3-

actinin or actin. Assuming that the Z-protein represents a stage of purifi­

cation intermediate between the purified a-actinin and Ebashi's a-actinin,

it should be possible to observe a trend in amino acid composition with the

Z-protein having an amino acid composition intermediate between that of

purified Q-actinin and Ebashi's a-actinin. This is clearly the case for

his, arg, asp, thr, glu, pro, gly, ala, ile, leu, and tyr, and only the val

composition of the Z-protein seems out of place. Thus, the data in Table 5

lend considerable support to the contention that DEAE-cellulose chromato­

graphy results in purification of a-actinin, and that a-actinin is a new

protein component of the myofibril, separate from denatured G-actin.

It was mentioned earlier that large amounts of phosphorylase were

present in the fraction of Seraydarian's (1967) crude a-actinin ex­

tracts. The composition of this fraction was somewhat variable, but its

estimated composition would be approximately 60 per cent 8.5S and 13S

(phosphorylase), 10 per cent 6S (CC-actinin), and 30 per cent unknown

species (large aggregates possibly including some denatured G-actin).

Thus, comparison between this uncharacterized fraction and any individual

protein can only be speculative in nature. Realizing this, there exists a

clear similarity between the amino acid composition of the fraction

89

and purified phosphorylase. Furthermore, it is clear that the amino acid

composition of purified a:-actinin differs from that of purified phosphorylase,

with purified OSactinin being higher in ser, glu, and ala, and lower in

lys, arg, thr, val, ile, tyr, and phe than phosphorylase.

A study on the stoichiometry of the interaction between actin and

purified a~actinin (DEAE) is shown in Figure 24. In this experi­

ment, CC-actinin was added in increasing amounts to a fixed amount of F-actin

and the mixture centrifuged at 45,000 rpm for 1 hr. This will cause com­

plete sedimentation of F-actin and the a-actinin-F-actin complex but any un­

bound a-actinin will remain in the supernatant. Thus, up to the point that

F-actin is able to bind a-actinin, there should be no protein in the centri­

fugal supernatant, but beyond this point, the excess a-actinin should appear

in increasing amounts in the supernatant. An extrapolation of the amounts

of protein appearing in the supernatant at these higher a-actinin to F-

actin ratios back to a supernatant protein concentration of zero should

give the stoichiometry of the a-actinin-F-actin interaction. In this ex­

periment, the protein left in the supernatant (after sedimentation of the

F-actin-a-actinin complex) was also assayed in the ATPase and turbidity

tests for a-actinin activity to provide conclusive evidence that the super­

natant protein was indeed a-actinin. It was found that a small amount of

F-actin (less than 5 per cent of the total) was left in the supernatant

when sedimented by itself and this amount was subtracted from the total mg

of protein in the supernatant for each tube. However, as a result of this

circumstance, there was always a small amount of protein present in the

supernatants, even before reaching the saturation point, and it was there­

fore possible to assay supernatant protein from tubes containing a-actinin

Figure 24. Stoichiometry of the a-actinin (DEAE) -F-actin interaction

Data points are the amount of total Pi5_25 (DEAE) left in the supernatant and not bound by F-actin or sedimented by itself. The dashed line represents the "expected" line or result if all a-actinin above a 41 per cent binding ratio did not interact with F-actin and was left free in the supernatant.

i 49.3% J NONSPECIFIC / OR WEAK ' BINDING /

UJ CO

Q

m

50.7% UNBOUND

o

20 30 40 50 60 70 80 90 100 110

ce-ACTININ ADDED AS PERCENT OF F-ACTIN

92

to F-actin ratios below the saturation point. Furthermore, a small propor­

tion of the a-actinin (about 10-15 per cent in these experiments) was sedi-

mented at 45,000 rpm for 1 hr even in the absence of any F-actin. There­

fore, a set of a-actinin "controls", containing a-actinin, but no F-actin,

was included in each experiment, and the amount of a-actinin sedimented in

the absence of F-actin was subtracted from the total amount of a-actinin

added to each tube. The data in Figure 24 show that F-actin will bind all

a-actinin added up to 41 per cent of its weight. Somewhat surprisingly,

some binding of a-actinin also occurs above this point, since if all the

a-actinin above 41 per cent of the F-actin present had been left free in

the supernatant, amounts of protein specified by the dotted line should

have been observed in the supernatant. Clearly, some "nonspecific or weak

binding" occurs above the 41 per cent binding ratio. There is no informa­

tion on the exact nature of this binding. It may be due to the presence of

two types of binding sites on F-actin or possibly to some kind of weak

interaction between individual a-actinin molecules.

The a-actinin assays on the supernatant protein confirmed the expecta­

tion that all the added a-actinin was bound below a-actinin-F-actin ratios

of 0.41. The. supernatant protein from the F-actin control (0 per cent) and

the 10 per cent and 20 per cent a-actinin tubes gave no response in the

turbidity assay for a-actinin activity (Figure 25). Supernatant protein

from the 30 per cent a-actinin tube did appear to give a slight activation

in the turbidity assay. This may suggest that the a-actinin-F-actin inter­

action is slightly dissociated near the saturation point, a conclusion sub­

stantiated by the slightly increased protein content in the supernatant from

the 30 per cent a-actinin tube (Figure 24). As shown in Figure 26, all

Figure 25. Effect of supernatant protein (Ql-actinin not bound by F-actin or sedimented by itself, Figure 24) on Mg"*"*"-activated acto-myosin superprecipitation

The per cent figures on the graph refer to the supernatant pro­tein that was obtained from the individual tubes containing the stated per cent or ratio of o;-actinin to F-actin. Final con­centrations for superprecipitation assay: 1 mM Mg"*"*", 1 mM ATP, 0.05 mM Ca"*"*", 100 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.4 mg actomyosin/ml. Supernatant proteins or control a-actinin was added as 5 per cent of actomyosin present. Temp., 26°C.

Figure 26. Effect of supernatant protein (a-actinin not bound by F-actin or sedimented by itself. Figure 24) on Mg^-activated acto­myosin superprecipitation

The per cent figures on the graph refer to the supernatant pro­tein that was obtained from the individual tubes containing the stated per cent or ratio of a-actinin to F-actin. Final con­centrations for superprecipitation assay: 1 mM Mg"*"*", 1 mM ATP, 0,05 mM Ca^, 100 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.4 mg actomyosin/ml. Supernatant proteins or control a-actinin was added as 5 per cent of actomyosin present. Temp.,26°C.

94

LU .5

CONTROL

Ê G C

O (O

2 .6

ë .5 z m oc o (/) m <

18 20 22 24 26 28 30 32 34 36

TIME (MIN.)

^15-25

(DEAE)

80% 0%

CONTROL

18 20 22 24 26 28 30 32 34 36

TIME (MIN.)

95

aupernatants tested above the 41 per cent binding ratio were highly active

in the turbidity test for a-actinin activity, indicating the presence of

0!-actinin in these supernatants. The results of the ATPase assay for

a-actinin activity in the supernatant fractions confirmed the turbidity

results. Supernatant protein from tubes with a-actinin to F-actin ratios

II' of 0.3 or lower caused no increase in the Mg -modified actomyosin ATPase

activity, but supernatant protein from tubes above this ratio produced an

increase in ATPase activity similar to that observed for a-actinin alone

(Figure 27) .

Only three previous studies have attempted to measure the stoichio-

metry of the a-actinin-F-actin interaction, lîriskey e^ al. (1967) used

analytical ultracentrifugation and measurements of peak height and arrived

at a binding ratio of 0.9 part of a-actinin to 1 part of F-actin by weight.

Unfortunately, this study suffered from two serious shortcomings: 1)

Briskey's a-actinin preparations were very heterogeneous and probably con­

tained less than 20 per cent of their protein as the 6S species, and 2)

the analytical ultracentrifugal method used was a very imprecise way to

study the interaction. Thus, Briskey's findings were later found in error

by Goll e^ (1969), but even these workers, who used a more purified

form of a-actinin and a different method, could not unequivocally determine

the stoichiometry of the a- ac tin in - F-actin interaction. The main problem in

this latter case still appeared to be the lack of homogeneity of the a-

actinin preparations. This problem of homogeneity also thwarted the efforts

of Drabikowski and Nowak (1968) to measure the binding ratio of a-actinin

to F-actin. These workers used "partially purified a-actinin" (10 gm) that

had been further purified by Nonomura's method (1967). One of the central

Figure 27. Effect of supernatant protein (cc-actinin not bound by F-actin or sedimented by itself. Figure 24) on activated actomyosin ATPase

The abscissa represents the supernatant protein that was obtained from the individual tubes containing the stated per cent or ratio of a-actinin to F-actin. Final concentra­tions: 1 mM Mg , 1 mM ATP, 0.05 mM Ca"*"^, 50 mM KCl, 20 mM Tris-acetate, pH 7.0, 0.2 mg actomyosin/ml. Supernatant proteins or control a-actinin was added to the extent of 20 per cent of the actomyosin present. Temp., 25°C.

.50-

.48

m

> .46

o < o» E

.44

(/) UJ

.42

40

i

/

CONTROL ACTOMYOSIN

± ± X _L X 10 20 30 40 50 60

cK- ACTININ ADDED AS PERCENT

/ ® CONTROL-ONLY oi-

ACTININ PRESENT

_J I I 80 90 100

F- ACTIN

98

difficulties in all these studies of the «-actinin-F-actin interaction is

that they require sizable amounts of purified a-actinin, which are diffi­

cult to obtain and cannot be stored for long periods of time.

A limited study was conducted on the possible relationship of a-actinin

to the Z-line. Stromer _et £l. (1967, 1969) had previously shown that

2-lines could be removed from "teased" glycerinated rabbit psoas fibers by

extraction with a low ionic strength solution for 10-14 days. Since this

extract obviously should contain the Z-line protein or proteins, the super­

natant solution from this extract was decanted, concentrated, and then

studied to determine Wiether it contained 0!-actlnin-like proteins. Stromer

et al. (1967) had previously shown that a 40 per cent ammonium sulfate

fraction of this crude extract contained protein which could, under proper

ionic conditions, rebind to certain parts of the I band, including the

region formerly occupied by the Z-line. Therefore, the concentrated Z-line

extract was fractionated by ammonium sulfate precipitation between 0-20 per

cent saturation and 20-40 per cent saturation, and some of the properties

of these two fractions (P^ and ^q) were studied in an effort to

determine whether cc-actinin was present in Z-line extracts.

The sedimentation profile of two of the concentrated crude Z-line ex­

tracts examined in this study are shown in Figures 28a and b. It is ob­

vious that both extracts contain a substantial proportion of rapidly sedi-

menting aggregates as well as several other different protein species. The

and P__ fractions from the same extract shown in Figure 28b are U-ZU Zv-4U

shown in Figures 28c and 28d, respectively. While a very large leading

edge can be observed in the Pq fraction early in the run, there is a

distinct peak sedimentlng with an observed S value of 6.1. In the P„„

Figure 28a, b. Sedimentation of concentrated crude Z-line extract

Final concentrations: a) 2.45 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. b) 4.66 mg protein/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. Temp., 20°C.

Figure 28c, d. Sedimentation of the Pq and fractions from Z-line extracts

Final concentrations : c) 6.19 mg PQ_2Q/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. d) 3.74 mg P^Q ^p/ml, 100 mM KCl, 20 mM Tris-acetate, pH 7.0. Temp., 20°C.

MINUTES

CONCENTRATED CRUDE (o)

2-LINE EXTRACT (b)

Po-20 Z-LINE EXTRACT (c )

^20-40 Z- LINE EXTRACT (d)

AFTER REACHING 59,780 RPM

101

Table 6. Effect of Po_20 ^20-40 extract) on actomyosin ATPase

Per cent of fraction added

0 2 5 10 20

*0-20

50 mM KCl 0.375^ 0.414 0.418 0.414 0.449

125 mM KCl 0.053 0.061 0.068 0.054 0.058

^20-40

50 mM KCl 0.375 0.409 0.370 0.421 0.435

125 mM KCl 0.053 0.059 0.063 0.067 0.062

^Conditions of assay: 0.20 mg actomyosin/ml, 1 mM ATP, 1 mM MgClg, 0.05 mM CaCl2, 20 mM Tris-acetate, pH 7.0, 25°C; Po_20 P2O-4O indicated as per cent of actomyosin present.

^Figures are p moles Pi/mg actomyosin/min.

fraction there are clearly at least 2 peaks which sediment with observed S

values of 6.1 and 8.45. By taking into consideration the protein concentra­

tions used in this run, the 6S species in the Pg^ fraction is present in

proportions similar to that of the 6S species in the Pq fraction. Ali-

quots of the Pq gg and P^^ fractions were tested for their effect on the

turbidity response of actomyosin suspensions at low ionic strength (Figure

18). When added in large amounts (30 per cent of the actomyosin present)

there was a clear indication that both extracts contained «-actinin

(probably the 6.IS peaks), and that the Pq fraction may possess a

slightly higher proportion of a-actinin. These results were confirmed by

the ATPase assay for a-actinin activity (Table 6).

102

The magnitude of the response in both tests was similar or slightly

better than that normally obtained with "partially purified a-actinin"

(11,4 gm). This "partially purified a-actinin" fraction is, of course,

heterogeneous, and it is evident that both the Pq 1*20-40 f^^ctions

from the Z-line are also very heterogeneous. However, it is clear that

Z-line extracts do contain a-actinin, although this obviously does not

prove that this a-actinin originated from the Z-line,

The nature of the 8,45S protein in the fraction was not examined

in detail, but it is probably phosphorylase. It should be mentioned that

phosphorylase or extracts containing primarily 8.5S and 13S protein species

such as P„- ,n, Figure 2, or P„„ from an Ebashi and Ebashi (1965) ex-

tract does not bind to F-actin and is either totally inactive or even

slightly inhibitory in the regular ATPase and turbidity tests for a-actinin.

This was shown by first mixing a phosphorylase-containing fraction (Pg^ ̂ q)

with F-actin, in an experiment similar to that described in Figure 24, and

then sedimenting the F-actin. This caused removal of all traces of (X-

actinin in these fractions, but the 8.5S and 13S proteins could still be

seen in an analytical ultracentrifuge diagram of the supernatant solution.

When this supernatant solution was tested in the ATPase or turbidity assays,

it exhibited no activity, even at very high accessory protein to actomyosin

ratios.

A very limited study on Z-line reconstitution was done by using the

experimental and ultrastructural procedures of Stromer e^ al^. (1967, 1969).

Figure 29a shows the usual morphology of glycerinated rabbit psoas muscle

prior to Z-line extraction. All the customarily observed bands of normal

striated muscle are seen (Huxley, 1957). Note especially the prominent

Figure 29a. Glycerinated rabbit psoas muscle prior to Z-line extraction

Sample was fixed with glutaraldehyde and postfixed with osmium tetroxide. Sections were stained with uranyl acetate and lead citrate. Z, Z-line; M, M-line. X 23,000

Figure 29b. Typical morphology of teased glycerinated rabbit psoas fibers after extraction

Sample was extracted with 2 mM Tris-HCl, pH 7.6, 6 mM 2-mercapto-ethanol for 12 days at 0°C. Note removal of Z-line and M-line material. X 33,560.

104

Figure 30a. Structure of Z-line extracted rabbit psoas fibers after recon­stitution with fraction

Conditions during the 64-hr recombination period were 6,3 mg P23.4o/ml, 100 mM KCl, 5 mM CaCl 5 mM MgCl2, 2 mM Tris-HCl, pH 7.6, 6 mM 2-mercapto-ethanol, 0°C. T, tufts, X 22,240,

Figure 30b. Structure of Z-line-extracted rabbit psoas fibers after recon­stitution with c Tc fraction

Conditions during the 64-hr recombination period were 10.0 mg Pl5.25/nil, 100 mM KCl, 5 mM CaClg, 5 mM MgClg, 2 mM Tris-HCl, pH 7.6, 6 mM 2-mercapto-ethanol, 0°C. T, tufts; CB, cross-bridges; Z, Z-line. X 33,560.

106

107

Z-line and the clear M-line.

Extraction for 12 days with a 2 mM Tris-HCl, pH 7.6, 6 mM 2-mercapto-

ethanol solution results in complete removal of both Z-lines and M-lines

(Figure 29b). The lighter I band is no longer bisected by the Z-line and

the thin filaments appear to simply terminate where the Z-line has been re­

moved.

Figure 30a shows results of a recombination experiment where the

fraction from an Ebashi and Ebashi (1965) extract poor in a-actinin but rich

in phosphorylase was made 100 mM in KCl and then allowed to interact for

64 hr at 0°C with the Z-line extracted, teased fiber bundles. The most

prominent feature of this interaction is the appearance of numerous small

tufts (T) of material bound in the lateral third of the I-band or alterna­

tively in the middle of the thin filament. There is no indication of any

Z-line reconstitution. This then serves as another internal control.

Figure 30b shows the results of a recombination experiment in which

the crude P,^ fraction was made 100 mM KCl and then allowed to interact Ij-ij

for 64 hr at 0°C with Z-line extracted, teased bundles of rabbit psoas

fibers. The increased density of the entire I band after this treatment is

quite evident. Most of this density is due to cross-bridges formed be­

tween adjacent thin filaments. In addition, some material is bound as

tufts. There is also evidence of some increased density in the Z-line re­

gion where, prior to recombination, the Z-lines had been completely ex­

tracted. A moderate reconstitution of Z-lines has occurred. Previously,

Stromer e^ ̂ 1. (1969) had shown that fX-actinin as made by Ebashi and Ebashi

(1965) did not reconstitute or gave only a very slight indication of abil­

ity to reconstitute Z-lines in Z-line extracted fibers. The experiment

108

described here indicates that a-actinin prepared by extraction at 2°C will

interact strongly with the thin filaments and will restore Z-line material.

109

DISCUSSION

The results of this study indicate that cc-actinin can be prepared by

direct extraction of "swollen" myofibrils at low ionic strength. This new

procedure differs in several important aspects from the procedure described

by Ebashi and Ebashi (1965). These differences include: 1) The new myo­

fibril extraction is done entirely at 2°C xdiereas the last 10-12 hr of

Ebashi's extraction is done at 20-25°C. Although no systematic studies on

the temperature lability of a-actinin have been conducted, it is a well-

known fact that the other myofibrillar proteins are very labile at tempera­

tures above 10°C, and it may be expected that a-actinin is also sensitive

to higher temperatures. During the course of this study, it was noted that

cc-actinin appeared to lose some of its activity in the ATPase and turbidity

tests after storage for 7-10 days at 0°C. Thus, a-actinin prepared by this

new method has had less exposure to denaturing conditions than a-actinin

prepared according to Ebashi's procedure. 2) The procedure used in this

study produces an extract which contains a three-to-five-fold higher propor­

tion of its protein as the active 6S species. This is very important when

attempting further purification of these crude extracts by column chroma­

tography. 3) The new procedure uses direct extraction of myofibrils which

can easily be prepared in very large quantities. This facilitates the

preparation of larger amounts of purified a-actinin. Because of these

advantages, the development of this new extraction procedure has been an

important contribution to the ability to purify a-actinin. Until now,

Ebashi's (Ebashi and Ebashi, 1965) procedure was the only one available for

extraction of a-actinin.

110

One of the uncertainties concerning cc-actinin is the amount of Ci-

actinin in muscle. Normal yields of "partially purified a-actinin" (10 gm)

in this study were about 360 mg protein from 200 gm of ground muscle. This

agrees with the yields reported by Goll et ^l. (1969). Assuming this

preparation contains approximately ten per cent of the 6S species (as

determined by area under the 6S-containing peak in the DEAE-cellulose

chromatogram), this amounts to approximately 36 mg 6S/200 gm muscle or

0.18 mg cc-actinin/gm muscle. The procedure developed in this study for the

^15 25 ff*ction yields about 214 mg protein from 200 gm ground muscle. This

is about 30 per cent 6S species, again as determined by area under the

6S-containing peak in the DEAE-cellulose chromatogram. Thus, there are

about 64.2 mg 6S/200 gm muscle or about 0.32 mg a-actinin/gm muscle.

Therefore, the method developed in this study has the fourth advantage of

a higher yield of active 6S species. There is, however, sensible agree­

ment between the two methods in yield of the 6S species. Both yields are

considerably lower than the yields of 10-15 mg a-actinin/gm of muscle re­

ported by Ebashi and Ebashi (1965) and are about equivalent to the yields

of 0.05-0.15 mg/gm of muscle that Goll £t £l. (1969) reported for A- and

Z-proteins. Assuming that muscle contains approximately 0.5 mg of a-

actinin/gm of fresh muscle and that about 20 per cent of total myofibrillar

protein is actin, it may be calculated that the ratio of a-actinin to

F-actin in muscle is about 1/40, by weight. Further, by accepting the

dimensions of the thin (actin) filament given by Hanson and Lowy (1963),

and by postulating that four a-actinin molecules with a molecular weight of

150,000 daltons each are required to bind one F-actin strand, 1 p in length,

to the Z-line, it is possible to estimate that there should be 1/36 as much

Ill

a-actlnin as F-actin in muscle. Thus, our yields of a-actinin are in good

agreement with the suggestion that a-actinin binds F-actin strands across

the Z-line.

It was discovered during the course of this study that the "swollen"

myofibrils may be "shrunk" by the addition of KCl. Although this lowers

the force needed to sediment the myofibrils and substantially increases the

volume of crude extract obtained, very little OSactinin can be found in

such an extract. It appears that addition of KCl causes the solubilized

a-actinin to rebind to the myofibrils, and it was therefore necessary to

use high speed centrifugation to obtain a-actinin from the "swollen" myo­

fibrils.

A substantial amount of phosphorylase activity can be found in crude

cc-actinin extracts prepared by either the new procedure or Ebashi's method.

As judged by ultracentrifugal methods and yields of the ^25-40 f^'&ction in

this study and the fraction from an Ebashi extract, much more phos­

phorylase (10-15 times) is present in extracts obtained by the procedure of

Ebashi and Ebashi (1965) than in extracts obtained by the new procedure.

The presence of phosphorylase, a sarcoplasmic enzyme, in the crude a-actinin

extracts is surprising since both the myofibrils used in this study and the

muscle residue used for Ebashi's cc-actinin extraction were thoroughly

washed and should not have contained any sarcoplasmic proteins. Apparently,

however, phosphorylase is not extracted very rapidly at temperatures of

0-4°C, and significant amounts of phosphorylase will remain with the myo­

fibrillar residue even though it is washed extensively.

The first goal of this study was the preparation of purified a-actinin

in quantities of several hundred mg or larger. This goal has been largely

112

achieved by the application of DEAE-cellulose chromatography to crude

a-actinin preparations extracted at 2°C. With this technique it has been

possible to prepare 100-200 itig of purified a-actinin by using a single pass

through a 2.5 by 25 cm DEAE-cellulose column. Purity of a protein prepara­

tion is always open to question and improvement with the development and

application of new techniques. Thus, it is likely that even the purified

a-actinin prepared in this study is not homogeneous in the ultimate sense,

but it is certainly a great deal less heterogeneous than any preparation

described thus far in the literature. Except for a small 9.IS peak, which

may be an aggregate of the 6S a-actinin molecule, very little heterogeneity

was evident in the purified a-actinin preparation when examined in the

analytical ultracentrifuge. Chromatography of the purified preparations on

either DEAE-cellulose or Sepharose 4B (4 per cent agarose) produced a single

symmetrical peak in the elution profile. Although there was some evidence

that rechromatography on DEAE-cellulose removed a very small amount of large

aggregates from the purified preparations, this additional purification is

probably more than offset by loss in specific activity of the 6S molecule

with increasing time of preparation. The species of large aggregates suc­

cessfully separated from the 6S species in this study was devoid of any

a-actinin activity.

This is the first reported attempt to use column chromatography to

purify a-actinin. Earlier investigations used repeated salting out or dif­

ferential sedimentation of large aggregates in the preparative ultracentri-

fuge for a-actinin purification. Since it has been suggested that a-actinin

is a structural protein, making up the Z-disk in the myofibril, it was not

clear before this study that any attempted purification of the a-actinin

113

molecule would be successful unless done in the presence of denaturing

solvents to prevent aggregation. It is now evident, however, that the 6S

species does not exhibit the marked aggregation tendencies characteristic

of many structural proteins and that the large aggregates found in crude

a-actinin preparations are not simply aggregated o;-actinin but instead are

probably denatured actin. This contention is supported by the amino acid

analyses of purified a-actinin, Ebashi's crude a-actinin, and actin. Puri­

fied Cc-actinin has an amino acid composition clearly different from that of

actin. Ebashi's crude CC-actinin has an amino acid composition intermediate

between that of purified a-actinin and actin. The Z-protein, described by

Coll e^ £l. (1969) as a partially purified form of Ebashi's crude Q!-actinin,

has an amino acid composition intermediate between purified (X-actinin and

Ebashi's crude 0!-actinin. Thus, for those amino acids where purified

a-actinin clearly differs from actin, there is a gradation in composition

from actin to Ebashi's CC-actinin to the Z-protein to purified a-actinin.

This constitutes additional evidence that the DEAE-purified OSactinin is

the most homogeneous CX-actinin preparation described thus far. The striking

differences in amino acid composition between the purified a-actinin and

actin also clearly demonstrate that a-actinin is not simply an unusual form

of denatured actin, a question that could not be definitely answered before

now.

The nature of the small amount of 9. IS species in the purified OL-

actinin preparations has not been elucidated. It is not phosphorylase be­

cause 1) it has a higher rate of sedimentation than the 8.5S characteristic

of the dimeric form of phosphorylase, 2) it is not evident in sedimentation

diagrams of purified a-actinin in 1 mM KHCO^ whereas the 8.5S phosphorylase

114

species sediments separately from the 6S a-actinin species in this medium,

3) it is bound to F-actin whereas the 8.5S phosphorylase is not, 4) con­

siderably higher KCl concentrations are required to elute it from DEAE-

cellulose columns than are necessary to elute phosphorylase, and 5) the

purified CZ-actinin preparations do not possess any detectable phosphorylase

activity. Because of its elution properties and its sedimentation behavior

in low ionic strength solutions, the 9.IS species may be a dimeric form of

the 6S a-actinin species.

The availability of purified a-actinin made it possible to success- .

fully determine the stoichiometry of the interaction between a-actinin and

F-actin. Previous attempts to do this have failed because only 10-20 per

cent of the protein in the a-actinin preparations then available would bind

to F-actin. Within the limits of experimental error (- 10 per cent), it

was found that all of the protein in the purified a-actinin preparations

would bind to F-actin. The results showed that F-actin would quantitatively

bind all a-actinin present up to 41 per cent of its own weight. About half

of the a-actinin added past this point was also bound to F-actin in some

type of "weak or non specific" binding. The nature of this weaker binding

is not known. By assuming a molecular weight of 60,000 daltons for the

G-actin subunits in F-actin and a molecular weight of 150,000 daltons for

the 6S species of a-actinin, it can be calculated that the stoichiometry of

0.4 parts of a-actinin to 1 part of F-actin by weight constitutes a molecu­

lar binding ratio of 1 molecule of a-actinin to 1 G-actin subunit. Thus,

a-actinin may bind to purified F-actin all along the length of the F-actin

strand.

Goll e^ a]^. (1967, 1969) and Masaki e^ (1967) have presented two

115

independent lines of evidence that CC-actinin is located in the Z-line of

the muscle fibril. By the use of Stromer's (1967, 1969) technique for first

extracting and then reconstituting the Z-lines in "teased" glycerinated

fiber bundles, it was possible, in this study, to obtain some additional

evidence on the presence of a-actinin in the Z-line. Z-line extracts after

concentration clearly possessed CK-actinin. This was shown by three differ­

ent tests: 1) a protein with the 6.IS value characteristic of a-actinin

was present in ammonium sulfate fractions of the Z-line extract, 2) the

Z-line extract exhibited the ability to increase the Mg^-modified ATPase

activity of an actomyosin suspension in a manner similar to that observed

for "partially purified a-actinin" preparations, 3) the Z-line extract had

the ability to accelerate the turbidity response of an actomyosin suspen­

sion at low ionic strength. The sedimentation diagrams also indicated the

presence of a substantial amount of large aggregates together with an 8.45S

protein (probably phosphorylase) in the concentrated crude extracts.

Previous characterization of the crude Z-line extracts by Stromer et al.

(1967, 1969) showed that tropomyosin was present, but that only a fraction

salting out between 0 and 40 per cent ammonium sulfate saturation would

cause any rebinding to the Z-line area. This fraction was shown to be

devoid of tropomyosin, which had previously been thought to make up the

Z-line, but tests for the presence of a-actinin in this fraction were not

done. In the present study, the crude Z-line extracts were fractionated

between 0-20 per cent (Pq ^g) and 20-40 per cent (Pgg ^q) ammonium sulfate

saturation and it was found that the Pq ^20 40 ff&ctions contained

a-actinin. However, it was not possible to do any further characterization

or purification of the Z-line extracts because of the very small amount of

116

protein available in such extracts, and because It was technically very dif­

ficult to obtain quantities of "teased" myofibrils large enough to produce

larger amounts of protein.

Although these studies clearly showed that C-actinin was present in

the Z-line extracts, this does not prove that the a-actinin originated from

the Z-line. To obtain further evidence on this point, attempts were made

to reconstitute Z-lines in Z-line-extracted fibrils. The limited experi­

ments done in this area suggested that CH-actinin may be a primary consti­

tuent of the Z-line. It was possible to obtain moderate reconstitution of

the Z-line by incubation of Z-line-extracted fibers with the crude

fraction from the 2°C-a-actinin extracts. This same fraction also caused

extensive formation of cross-bridges in the 1 band region. It was obvious

that incubation of the Z-line-extracted fibers with the 25 fraction

caused much more binding in the Z-line region than was previously found by

Stromer £t al. (1969) when incubating the Z-line-extracted fibers with

a-actinin prepared according to the procedure of Ebashi and Ebashi (1965).

Incubation with Ebashi's o:-actinin appeared to result primarily in the

formation of cross-bridges in the extracted fibers. Neither the exact

nature of the cross-bridges nor the mechanism underlying binding to the

Z-line region is well understood. It was shown by Stromer ejt £l. (1969)

that incubation with Ebashi's extracts containing high protein concentra­

tions (> 10 mg/ml) was needed to cause any binding in the Z-line area. A

similar effect could be obtained with less than 5 mg/ml of the frac­

tion. This indicates that the active protein made up only a small propor­

tion of the total protein in the Ebashi extracts and that a higher protein

concentration of the 6S species favors binding to the Z-line area for steric

117

or other unknown reasons. It is probable that the extraction of tropomyosin

along the length of the thin filament, which occurs concomitantly with

Z-line extraction, uncovers sites on the actin filament which then bind

Ql-actinin. This may explain the formation of cross-bridges in the extracted

fibers. Whatever the mechanism underlying Z-line reconstitution, it is

clear that the 25 fraction, which contains a large proportion of the 6S

CX-actinin species and which has not been subjected to high temperatures

during extraction, causes some Z-line reconstitution.

One other interesting observation made during the Z-line reconstitu­

tion experiments was that incubation with the 25 fraction did not

cause reconstitution of the M-line in the extracted fibers. The conditions

for Z-line extraction from the glycerinated fibers also leads to complete

extraction of the M-lines. Stromer e^ a]^. (1969) found that incubation of

the extracted fibers with Ebashi's a-actinin did cause reconstitution of

the M-line. Thus, Ebashi's a-actinin preparation plainly possesses a pro­

tein species not contained by the P^^ fraction.

The evidence presented in this study together with the earlier evi­

dence of Goll e^ al. (1967, 1969) and Masaki ejt al. (1967) clearly places

a-actinin in the Z-line. In their original discovery of a-actinin, Ebashi

and Ebashi (1965) suggested that the function of a-actinin in muscle was to

promote and accelerate the actin-myosin interaction, particularly at high

(physiological) ionic strengths where this Interaction does not occur in

purified actomyosin systems vitro. However, when it was shown that

Ebashi's OSactinin preparations were not active under physiological condi-

++ tions (120-150 mM KCl, 5 mM ATP, 5-10 mM Mg ), a contraction-promoting

function for a-actinin seemed unlikely. Location of a-actinin in the Z-line

118

favored the idea that the role of cc-actinin in muscle is purely a structural

one, serving to connect actin filaments together across the Z-line. It is

difficult to understand how 0!-actinin located in the Z-line could affect

the actin-myosin interaction which occurs only at the region of overlap be­

tween the thick and thin filaments, some 0.8 p distant from the Z-line. On

the other hand, it was clear that addition of CK-actinin to ija vitro acto-

myosin suspensions did enhance and accelerate the contractile properties s

of these suspensions at low (0.02-.07) ionic strengths.

This study, however, has shown that the failure of earlier a-actinin

extracts to cause "contraction enhancement" at higher KCl (100-125 mM) con­

centrations was probably due either to the small amount of actual a-actinin

in these extracts, or to partial denaturation caused by room temperature

extraction. Thus, it was found in this study that purified a-actinin did

possess the ability to increase the ATPase activity of actomyosin suspen­

sions at physiological ionic strengths. In fact, the per cent increase in

ATPase activity caused by purified a-actinin was greater at 100-125 mM KCl

than at lower KCl concentrations. Furthermore, it was shown that addition

of purified a-actinin in sufficient quantities to an actomyosin suspension

at conditions approximating physiological conditions would cause an in­

stantaneous turbidity response. Thus, it is clear that purified a-actinin

does have "contraction enhancement" properties at high (physiological)

ionic strengths. The results of this study have also shown that the

accelerating effects of purified a-actinin are specific for contraction--H- ++

related processes. Thus, neither the Mg - nor Ca -modified ATPase activi­

ties of myosin alone are affected by a-actinin, and, of course, myosin

-H-alone does not give a contractile response. Moreover, the Ca -modified

119

ATPase activity of actomyosin suspensions is not affected by purified d-

actinln, and neither does the addition of Ca^ alone cause a contractile

response in actomyosin suspensions. Finally, purified a-actinin does not

appear to increase the ITPase activity of actomyosin suspensions, and it has

been shown that ITP will produce only a weak contractile response in myo-

-H- ++ fibrils. It is only the Mg -modified ATPase activity and the Mg -modified

turbidity response of actomyosin suspensions that are accelerated by puri­

fied CC-actinin, and it is generally accepted that these two activities are

closely related to the vivo contraction process.

In addition to these considerations, purification of a-actinin has

made it possible to show that very small amounts of purified a-actinin (5

per cent or less of the actomyosin present) will cause clear and reproduci­

ble increases in the ATPase activity and turbidity responses of actomyosin

suspensions at 100 mM KCl. Therefore, the previous reports (Maruyama,

1966a; Seraydarian e^ , 1967) indicating that very high ratios of a-

actinin to actomyosin (up to 2 to 10:1) were necessary to cause any in­

crease in ATPase activity or turbidity response at 100 mM KCl were probably

due to the low purity of the a-actinin preparations used.

Although there is a substantial amount of evidence suggesting that

a-actinin does modify the actin-myosin interaction, there is no clear in­

dication of how this modification occurs. This study has provided further

evidence that a-actinin is located in the Z-line and that purified a-actinin

interacts only with F-actin. It is possible that addition of a-actinin to

actomyosin suspensions causes cross-linking of actin filaments and results

in a steric arrangement which favors interaction with myosin. Thus, the

"contraction enhancement" properties of a-actinin may be simply secondary

120

effects of its primary structural role of cross-linking actin filaments.

If a-actinin does have a direct effect on the molecular transduction

process of contraction, the effect must involve long-range changes of a

type not yet familiar in biology.

Whatever the final judgment on the role of CX-actinin in muscle, it is

probable that the turbidity and ATPase assays will remain useful tests for

the presence of a-actinin. In fact, if it develops that o:-actinin has

primarily a structural function in muscle, it will be somewhat ironical

that the most useful tests for the presence of this structural protein will

involve assays of a "biological activity". A similar situation has occurred

with tropomyosin, a myofibrillar protein whose primary role ostensibly is

to act as a "cement", binding troponin to the actin filament. Until Schaub

et al. (1967) showed that tropomyosin would decrease the Ca^-modified

ATPase activity of actomyosin suspensions at ionic strengths below 0.02,

it was practically impossible to assay for the presence of small amounts of

tropomyosin. The same situation may exist with a-actinin, except that in

this case, the "biological assay" was discovered first.

121

SUMMARY

A new procedure has been developed for the preparation of a crude

Ot-actinin extract. The extract is obtained by preparing myofibrils, wash­

ing them several times in water until "swollen", adjusting the pH to 8.5,

and allowing the low ionic strength "swollen" myofibril suspension to stand

for 2-3 days at 2°C. The crude a-actinin extract (2°C-a-actinin) is subse­

quently collected by sedimenting the "swollen" myofibrils at high centri­

fugal force, and decanting the clear supernatant solution.

The crude 2°C-a-actinin extract has been fractionated with ammonium

sulfate into several fractions. The fraction salting out between 15 and 25

per cent ammonium sulfate saturation, the fraction, was found to

contain 30-35 per cent of its protein as the 6S a-actinin species. Thus,

this fraction was a much better starting point for further purification

procedures than CC-actinin made by the conventional procedures of Ebashi and

Ebashi (1965) and Seraydarian e^ al. (1967).

The P-c fraction has a three-to-five-fold higher specific activity

in the ATPase and turbidity assays of a-actinin activity than did "partially

purified a-actinin" preparations. Moreover, in contrast to previous re­

ports on "partially purified a-actinin" (Seraydarian e_t £l. (1967), the

^15 25 ff&ction exhibited CC-actinin activity at KCl concentrations near the

physiological range. It was shown that the ^5 fraction did not have

++ 44-any effect on the Mg - or Ca -modified ATPase activities of myosin alone,

44. or on the Ca -modified'ATPase activity of actomyosin. Rather, only the

++ ++ Mg -modified ATPase activity and the Mg -modified turbidity response of

actomyosin suspensions were accelerated by addition of the fraction.

122

Purification of the 25 fraction was accomplished by using either

molecular exclusion or cellulose ion-exchange chromatography. The largest

single kind of contaminant in the P^^ 25 fraction was a class of large

aggregates. Neither Sephadex-200 nor Sagavac 8F (8 per cent agarose)

provided sufficient separation between these large aggregates and the 6S

a-actinin species and were, therefore, not very effective for purification

of the 6S species. Long (190 cm) columns of Sepharose 4B (4 per cent

agarose) did give more separation between the large aggregates and the 6S

species, but even after chromatography on Sepharose 4B, analytical ultra-

centrifugation indicated the presence of a few large aggregates in the 6S-

containing fractions.

DEAE-cellulose chromatography was the most effective method of separat­

ing the 6S species from the large aggregates. The 6S a-actinin species

was eluted from the DEAE-column by 250-300 mM KCl, but the large aggregates

were very tightly bound and required 0.5 N NaOH for elution. Since the

DEAE-cellulose columns could effectively purify five-to-six-fold larger

quantities of protein, and much shorter times were required for elution of

the 6S peak from DEAE-columns, DEAE-cellulose is the preferred method for

the purification of a-actinin fractions. a-Actinin purified by DEAE-

cellulose chromatography exhibited two to three times higher specific

activity in the turbidity test than did the original 25 fraction.

Rechromatography of DEAE-purified o:-actinin on either Sepharose 4B or

a second DEAE-cellulose column resulted in very little additional purifica­

tion and confirmed the conclusion that a-actinin that had been purified by

a single pass through a DEAE-cellulose column was nearly homogeneous.

Analytical ultracentrifugal studies of DEAE-purified a-actinin revealed the

123

presence of two peaks, a main peak of 6.23S and a small peak of approxi­

mately 9.IS. The 9.IS may be an aggregate of the 6.28 a-actinin peak since

it is not present in sedimentation diagrams in low ionic strength solutions.

Studies on the DEAE-purified a-actinin showed that purified a-actinin

was much more susceptible to tryptic hydrolysis than the "partially puri­

fied a-actinin" preparations formerly studied. Forty min of trypsin treat­

ment at trypsin-to-purified a-actinin ratios of 1:50 by weight caused the

appearance of two peaks in the analytical ultracentrifuge, a small broad

2.6S peak and a larger 4.7S peak.

The amino acid composition of DEAE-purified a-actinin was clearly dif­

ferent from the amino acid composition of actin. This demonstrates that

a-actinin is a new protein component of the myofibril and not simply an

unusual form of denatured actin, a fact that could not be decided before

now.

The availability of purified a-actinin also made it possible for the

first time to ascertain the stoichiometry of the a-actinin-F-actin inter­

action. This stoichiometry was found to be 0.41 parts of a-actinin to 1

part of F-actin, by weight. This weight ratio corresponds very closely to

a molecular binding ratio of one a-actinin molecule for each G-actin sub-

unit in the F-actin filament.

Analytical ultracentrifugation of DEAE-purified a-actinin in denaturing

solvents revealed the presence of two boundaries in such solvents. Either

the 6S a-actinin molecule has a heterogeneous subunit composition or the

DEAE-purified a-actinin is not completely homogeneous.

It was demonstrated by analytical ultracentrifugation, and by use of

the ATPase and turbidity assays, that a-actinin was present in low ionic-

124

strength extracts of "teased" glycerinated rabbit psoas fibers. This low

ionic-strength extraction causes removal of the Z- and M-lines from the

glycerinated fibers. Incubation of the Z-line-extracted fibers with the

^15 25 O^actinin fraction in 100 mM KCl resulted in some binding of material

to the Z-line region as well as extensive cross-bridge formation between

adjacent thin filaments. Thus, it has been shown that a-actinin is present

in Z-line extracts of muscle fibrils, and that incubation with a protein

fraction rich in a-actinin will cause moderate reconstitution of the Z-line

structure in Z-line-extracted fibrils.

125

CONCLUSIONS

As a resuit of this study, the following conclusions seem justified:

1. a-Actinin can be extracted directly from "swollen" myofibrils at

2°C. This procedure has several advantages over the existing procedure for

extracting a-actinin because: a) it yields an extract containing a higher

proportion of the active èS CC-actinin species, b) it is done at 2°C, thereby

preventing any denaturation of a-actinin due to exposure to room tempera­

ture, and c) it is easily adapted to the preparation of large amounts of

CC-actinin because of the ease of making large quantities of myofibrils.

2. A fraction, can be obtained from the myofibrillar CC-actinin

extract by ammonium sulfate fractionation between 15-25 per cent satura­

tion. This fraction contains a three-to-five-fold higher proportion of the

6S-o;-actinin species and exhibits a three-to-five-fold higher specific

activity in the ATPase and turbidity tests for a-actinin than do other

"partially purified a-actinin" preparations. The largest per cent increase

in ATPase activity caused by the P^^ fraction occurs at 100-125 mM KCl.

Thus, in contrast to "partially purified a-actinin" preparations, the

^15 25 f^&ction exhibits a-actinin activity at conditions very close to

physiological conditions.

3. The 25 fraction can be purified by using chromatography on

either Sepharose 4B or DEAE-cellulose. DEAE-cellulose columns effected the

most nearly complete separation of the 6S a-actinin' species from the large

contaminating aggregates normally present in the P^^ fraction. A major

6S a-actinin peak and a small 9.IS peak, possibly an aggregate of 6S a-

actinin species, are the only species visible in analytical ultracentrifugal

126

patterns of the DEA.E-purifled a-actinln.

4. Denaturing solvents cause the appearance of two peaks in analytical

ultracentrifugation diagrams of purified a-actinin. Either a-actinin has a

subunit structure or some heterogeneity still exists in the DEAE-purified

preparations.

5. Purified CX-actinin is more susceptible to proteolytic hydrolysis

by trypsin than unpurified Qi-actinin extracts. A heterogeneous 2.6S and a

major 4.7S species are produced by 40 min of trypsin treatment at trypsin

to purified a-actinin ratios of 1:50 by weight.

6. Purified a-actinin has an amino acid composition clearly distinct

from that of any other known myofibrillar protein. Thus, O-actinin is not

merely some unusual form of denatured G-actin, a fact that remained in

question until now.

7. The binding ratio of purified a-actinin to F-actin at 0°C is 0.41

parts of a-actinin to 1 part of F-actin by weight. This corresponds to a

molecular binding ratio of one a-actinin molecule to each G-actin subunit

in the F-actin filament.

8. a-Actinin is present in low ionic-strength extracts of "teased"

myofibril bundles. Electron microscopic examination shows that this low

ionic-strength extraction causes removal of the Z- and M-lines from the

fiber bundles.

9. Incubation with the P,^ oc a-actinin fraction in 100 mM KCl causes

a moderate amount of binding to the Z-line region, of the Z-line-extracted

fibril bundles. Thus, fractions rich in a-actinin will cause moderate re-

constitution of Z-lines.

127

LITERATURE CITED

Appleman, M. M., A. A. Yunis, E. G. Krebs, and E. H. Fischer. 1963. Com­parative studies on glycogen phosphorylase. V. The amino acid compo­sition of rabbit and human skeletal muscle phosphorylase. J. Biol. Chem. 238:1358-1361.

Bailey, K. 1948. Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem. J. 43:271-279.

Banga, I. and A. Szent-Gyorgyi. 1943. The influence of salts on the phos­phatase action of myosin. Univ. Szeged Inst. Med. Chem. Stud. 3:72-75.

Briskey, E. J., K. Seraydarian, and W. F. H, M. Mommaerts, 1967a. The modification of actomyosin by a-actinin. II. The effect of a-actinin upon contractility. Biochira. Biophys. Acta 133:412-423.

Briskey, E. J., K. Seraydarian, and W. F. H. M. Mommaerts. 1967b. The modification of actomyosin by a-actinin. III. The interaction between cc-actinin and actin. Biochim. Biophys. Acta 133:424-434.

Carsten, M. E. 1963. Actin, its amino acid composition and its reaction with iodoacetate. Biochemistry 2:32-34.

Chaplain, R. A. 1967. Thermal motion as the rate-limiting step in the superprecipitation of actomyosin gels. Arch. Biochem. Biophys. 122: 518-519.

Corsi, A., V. Muscatello, and I. Ronchetti. 1967. Electron microscope ob­servations of the location of actin and tropomyosin in the rabbit myo­fibril. J. Ultrastructure Res. 19:260-272.

Corsi, A. and S. V. Perry. 1958. Some observations on the localization of myosin, actin and tropomyosin in the rabbit myofibril. Biochem. J. 68:12-17.

Drabikowski, W., Y. Nonomura, and K. Maruyama. 1968. Effect of tropomyo­sin on the interaction between F-actin and the 6S component of oc-actinin. J. Biochem. 63:761-765.

Drabikowski, W. and E. Nowak. 1968. The interaction of n^actinin with F-actin and its abolition by tropomyosin. European J. Biochem. 5:209-214.

Ebashi, S. 1960. Calcium binding and relaxation in the actomyosin system. J. Biochem. 48:150-151.

Ebashi, S. 1961. Calcium binding activity of vesicular relaxing factor. J. Biochem. 50:236-244.

128

Ebashi, S, 1963. Third component participating in the superprecipitation of "natural actomyosin". Nature 200:1010.

Ebashi, S. and F. Ebashi. 1965. a-Actinin, a new structural protein from striated muscle. I. Preparation and action on actomyosin-ATP inter­action. J. Biochem. 58: 7-12.

Ebashi, S., F. Ebashi, and A. Kodama. 1967. Troponin as the Ca"^-receptive protein in the contractile system. J. Biochem. 62:137-138.

Ebashi, S., F. Ebashi, and K. Maruyama. 1964. A new protein factor pro­moting contraction of actomyosin. Nature 203:645-646.

Ebashi, S, and M. Endo. 1964. Further studies on the calcium-binding activity of the relaxing factor. In J. Gergely, ed. Biochemistry of muscle contraction. Pp. 199-206. Little, Brown and Company, Boston, Massachusetts.

Ebashi, S. and A. Kodama. 1965. A new protein factor promoting aggrega­tion of tropomyosin. J. Biochem. 58: 107-108,

Ebashi, S. and A. Kodama. 1966. Interaction of troponin with F-actin in the presence of tropomyosin. J. Biochem. 59:425-426.

Ebashi, S., A. Kodama, and F. Ebashi. 1968. Troponin. I. Preparation and physiological function. J. Biochem. 64:465-477.

Ebashi, S. and F. Lipman. 1962. Adenosine triphosphate-linked concentra­tion of calcium ions in a particulate fraction of rabbit muscle. J. Cell Biol. 14:389-400.

Eisenberg, E. and C. Moos. 1967. The interaction of actin with myosin and heavy meromyosin in solution at low ionic strength. J. Biol. Chem. 242:2945-2951.

Endo, M. 1964. The superprecipitation of actomyosin and its ATPase activity in low concentration of ATP. J. Biochem. 55:614-622.

Endo, M., Y. Nonomura, T. Masaki, I. Ohtsuki, and S. Ebashi. 1966. Locali­zation of native tropomyosin in relation to striation patterns. J. Biochem. 60:605-608.

Engelhardt, W. A. and M. N. Ljubimova. 1939. Myosin and adenosine-triphos­phatase. Nature 144:668-669.

Coll, D. E., W. G. Hoekstra, and R. W. Bray. 1964. Age-associated changes in bovine muscle connective tissue. I. Rate of hydrolysis by col-lagenase. J. Food Sci. 29:608-614.

129

Goll, D. E., W. F. H. M. Mommaerts, M. K. Reedy, and K. Seraydarian. 1969. Studies on a-actinin-like proteins liberated during trypsin digestion of «-actinin and of myofibrils, [To be published in Biochim. Biophys. Acta £a. 1969.]

Goll, D. E., W. F. H. M. Mommaerts, and K. Seraydarian. 1967. Is a-actinin a constituent of the Z-band of the muscle fibril? Fed. Proc. 26:499.

Goll, D. E. and R. M. Robson. 1966. Molecular properties of post-mortem muscle. I. Myofibrillar nucleosidetriphosphatase activity of bovine muscle. J. Food Sci. 32:323-329.

Gornall, A. G., C. T. Bardawill, and M. M. David. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: 751-766.

Hanson, J. and J. Lowy. 1963. The structure of F-actin and of actin fila­ments isolated from muscle. J. Molecular Biol. 6:46-60.

Huxley, H. E. 1953. Electron microscope studies of the organization of the filaments in striated muscle. Biochim. Biophys. Acta 12:387-394.

Huxley, H, E. 1957. The double array of filaments in cross-striated muscle. J. Biophys. Biochem. Cytol. 3:631-646.

Huxley, H. E. 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Molecular Biol. 7:281-308.

Katz, A. M. 1963. Some aggregation phenomena in non-polymerizing actin solutions. Biochim. Biophys. Acta 78:226-228.

Kominz, D. R. and K. Maruyama. 1967. Does native tropomyosin bind to myosin? J. Biochem. 61:269-271.

Leadbeater, L. and S. V. Perry. 1963. The effect of actin on the mag­nesium-activated adenosine triphosphate of heavy meromyosin. Biochem. J. 87:233-238.

Levy, H. M. and M. Fleisher. 1965a. Studies on the superprecipitation of actomyosin suspensions as measured by. the change in turbidity. I. Effects of adenosine triphosphate concentration and temperature. Biochim. Biophys. Acta 100:471-478.

Levy, H. M. and M. Fleisher. 1965b. Studies on the superprecipitation of actomyosin suspensions as measured by the change in turbidity. II. The relationship to hydrolysis of adenosine and inosine triphosphate. Biochim. Biophys. Acta 100:491-502.

130

Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

Maruyama, K. 1965. Some physico-chemical properties of p-actinin, "actin-factor" isolated from striated muscle. Biochim. Biophys. Acta 102: 542-548.

Maruyama, K. 1966a. Effect of actinins on the adenosinetriphosphatase activity of actomyosin at low ionic strength. J. Biochem. 59:422-424.

Maruyama, K. 1966b. Effect of p-actinin on the particle length of F-actin. Biochim. Biophys. Acta 126:389-398.

Maruyama, K. and S. Ebashi. 1965. a-Actinin, a new structural protein from striated muscle. II. Action on actin. J. Biochem. 58:13-19.

Maruyama, K. and J. Gergely. 1962a. Interaction of actomyosin with adenosine triphosphate at low ionic strength. I. Dissociation of actomyosin during the clear phase. J. Biol. Chem. 237:1095-1099.

Maruyama, K. and J. Gergely. 1962b. Interaction of actomyosin with adenosine triphosphate at low ionic strength. II. Factors influenc­ing clearing and superprecipitation: adenosine triphosphatase and birefringence of flow studies. J. Biol. Chem. 237:1100-1106.

Maruyama, K. and Y. Ishikawa. 1964. Effect of temperature and potassium chloride concentration on the onset of superprecipitation of actomyosin adenosine triphosphatase studies. J. Biochem. 55:110-115.

Maruyama, K. and M. Kawamura. 1968. Effect of H-meromyosin and P-actinin on the particle length of F-actin. J. Biochem. 64:263-265.

Masaki, T., M. Endo, and S. Ebashi. 1967. Localization of 6S component of a-actinin at Z-band, J. Biochem. 62:630-632.

Nonomura, Y. 1967. A study on the physico-chemical properties of a-actinin. J. Biochem. 61:796-802.

Ohtsuki, I., T. Masaki, Y. Nonomura, and S. Ebashi. 1967. Periodic distri­bution of troponin along the thin filament. J. Biochem. 61:817-819.

Perry, S. V. and A. Corsi. 1958. Extraction of proteins other than myosin from the isolated rabbit myofibril. Biochem. J. 68:5-12.

Perry, S. V. and T. C. Grey. 1956. A study of the effects of substrate concentration and certain relaxing factors on the magnesium-activated myofibrillar adenosine triphosphatase. Biochem. J. 64: 184-192.

131

Robson, R. M., D. E. Coll, and M. J. Temple. 1968. Determination of pro­teins in "Tris" buffer by the biuret reaction. Analyt. Biochem. 24; 339-341.

Schachman, H. K. 1959. Ultracentrifugation in biochemistry. Academic Press, Inc., New York, New York.

Schaub, M. C., S. V. Perry, and D. J. Hartshorne. 1967. The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin. Biochem. J. 105:1235-1243.

Seraydarian, K., E. J. Briskey, and W. F. H. M. Mommaerts. 1967. The modification of actomyosin by cc-actinin. I. A survey of experimental conditions. Biochim. Biophys. Acta 133:379-411.

Seraydarian, K., E. J. Briskey, and W. F, H. M. Mommaerts, 1968. The modification of actomyosin by CC-actinin. IV. The role of sulfhydryl groups. Biochim. Biophys. Acta 162:424-434.

Spicer, S. S. 1952. The clearing response of actomyosin to adenosine-triphosphate. J. Biol. Chem. 199:289-300.

Stromer, M. H., D. J. Hartshorne, H. Mueller, and R. V. Rice. 1969. The effect of various protein fractions on Z- and M-line reconstitution. J. Cell Biol. 40:167-178.

Stromer, M. H., D. J. Hartshorne, and R. V. Rice. 1967. Removal and re-constitution of Z-line material in a striated muscle. J. Cell Biol. 35:C23-C28.

Szent-Gyorgyi, A. 1951. Chemistry of muscular contraction. 2nd ed. Academic Press, Inc., New York, New York.

Tada, M. and Y. Tonomura. 1967. Superprecipitation of myosin B induced by ATP as a nucleated growth process. J. Biochem. 61:123-135.

Taussky, N. H. and E. Shorr. 1953. A microcplorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202:675-685.

Tokiwa, T., T. Shimada, and Y. Tonomura. 1967. Role of ADP of F-actin in superprecipitation and enzymatic activity of actomyosin. J. Biochem. 61:108-122.

Tonomura, Y., T. Tokiwa, and T. Shimada. 1966. Role of ADP bound to F-actin in superprecipitation and enzymatic activity of actomyosin. J. Biochem. 59:322-324.

Tonomura, Y. and J. Yoshimura. 1960. Inhibition of myosin B-adenosine-triphosphatase by excess substrate. Arch. Biochem, Biophys. 90:73-81.

132

Weber, A. 1959. On the role of calcium in the activity of adenosine 5'-triphosphate hydrolysis by actomyosin. J. Biol. Chem. 234:2764-2769.

Weber, A., R. Herz, and I. Reiss. 1964. Role of calcium in contraction and relaxation of muscle. Federation Proc. 23:896-900.

Weber, A. and S. Winicur. 1961. The role of calcium in the superprecipi-tation of actomyosin. J. Biol. Chem. 236:3198-3202.

Yasui, B., F. Fuchs, and F. N. Briggs. 1968. The role of the sulfhydryl groups of tropomyosin and troponin in the calcium control of acto­myosin contractility. J. Biol. Chem. 243:735-742.

Yasui, T. and S. Watanabe. 1965. A study of "superprecipitation" of myosin B by the change in turbidity. J. Biol. Chem. 240:98-104.

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ACKNOWLEDGEMENTS

The author expresses sincere appreciation to Dr. D. E. Goll for his

guidance, encouragement, and counsel throughout the course of the research

and in the preparation of the dissertation.

Special thanks go to Dr. N. Arakawa for the many suggestions that were

very helpful during this investigation.

The author expresses sincere appreciation to Dr. M. H. Stromer who

took the electron micrographs presented in this manuscript and for many

helpful suggestions.

The author is grateful to Dr. D. J. Graves for checking some of the

protein fractions mentioned in this manuscript for phosphorylase activity.

The author would also like to thank those members of his graduate com­

mittee: Dr. D. E. Goll, Dr. H. E. Snyder, Dr. D. J. Graves, Dr. P= E.

Outka, and Dr. F. C, Parrish for the guidance given during the course of

his graduate program.

The author is especially grateful to his wife Susan and daughter

Kristi. To Susan for her help in typing the original dissertation, and to

both for their patience, encouragement, and understanding.

Acknowledgement is also made of the financial support of a Predoctoral

Fellowship from the Division of General Medical Sciences, United States

Public Health Service.